Methods for Mass Spectrometry of Mixtures of Protein or Polypeptides Using Proton Transfer Reaction

ABSTRACT

Applications of ion-ion reaction chemistry are disclosed in which proton transfer reactions (PTR) are used to (1) simplify complex mixture analysis of samples introduced into a mass spectrometer, and (2) improve resolution and sensitivity for the analysis of large proteins in excess of 50 kDa by removing charge and reducing the collisional cross section.

TECHNICAL FIELD

The present invention relates to mass spectrometry and, moreparticularly, relates to methods for analyzing complex mixtures ofproteins or polypeptides by mass spectrometry of proton transferreaction product ions generated from proteins, polypeptides, and otherbiologically relevant multiply-charged species and the application ofthese methods to the identification and characterization ofmicroorganisms.

BACKGROUND ART

In recent years, mass spectrometry has gained popularity as a tool foridentifying microorganisms due to its increased accuracy and shortenedtime-to-result when compared to traditional methods for identifyingmicroorganisms. To date, the most common mass spectrometry method usedfor microbial identification is matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry. In MALDI-TOF,cells of an unknown microorganism are mixed with a suitable ultravioletlight absorbing matrix solution and are allowed to dry on a sampleplate. Alternatively, an extract of microbial cells is used instead ofthe intact cells. After transfer to the ion source of a massspectrometer, a laser beam is directed to the sample for desorption andionization of the proteins and time-dependent mass spectral data iscollected.

The mass spectrum of a microorganism produced by MALDI-TOF methodsreveals a number of peaks from intact peptides, proteins, proteinfragments, and other molecules that constitute the microorganism's“fingerprint”. This method relies on the pattern matching of the peakprofiles in the mass spectrum of an unknown microorganism to a referencedatabase comprising a collection of mass spectra for knownmicroorganisms obtained using essentially the same experimentalconditions. The better the match between the spectrum of the isolatedmicroorganism and a spectrum in the reference database, the higher theconfidence level in identification of the organism at the genus,species, or in some cases, subspecies level. Because the method reliesupon matching the patterns of peaks in MALDI-TOF mass spectra, there isno requirement to identify or otherwise characterize the proteinsrepresented in the spectrum of the unknown microorganism in order toidentify it.

Although MALDI-TOF methods are rapid and cost effective, they havelimitations that restrict the range of applications to pathogencharacterization and identification including but not limited tovirulence detection and quantitation, resistance marker determination,strain matching, and antibiotic susceptibility testing to name a few.The information content within a MALDI mass spectrum reflects the mostabundant and ionizable proteins which are generally limited to ribosomalproteins at the experimental conditions used. Because ribosomal proteinsare highly conserved among prokaryotes, differentiation of closelyrelated microorganisms by MALDI-TOF is limited. In this case many of theribosomal proteins across closely related species contain either thesame or slightly different amino acid sequences (i.e. single amino acidsubstitutions) that cannot be effectively differentiated with lowresolution mass spectrometers. Moreover, determination of strain and/orserovar type, antibiotic resistance, antibiotic susceptibility,virulence or other important characteristics relies upon the detectionof protein markers other than ribosomal proteins which further limitsthe application of MALDI-TOF for microbial analysis. Laboratories usingMALDI-TOF for identification of microorganisms must use other methods tofurther characterize the identified microbes. In addition, the MALDI-TOFmethod's reliance upon matching spectral patterns requires a pureculture for high quality results and thus is not generally suitable fordirect testing, mixed cultures, blood culture, or other complex samplescontaining different microorganisms.

Several other mass spectrometry methods for detection of microorganismshave been used. For example, mass spectrometry-based protein sequencingmethods have been described wherein liquid chromatography is coupled totandem mass spectrometry (LC-MS/MS) and sequence information is obtainedfrom enzymatic digests of proteins derived from the microbial sample.This approach, termed “bottom-up” proteomics, is a widely practicedmethod for protein identification. The method can provide identificationto the subspecies or strain level as chromatographic separation allowsthe detection of additional proteins other than just ribosomal proteins,including those useful for characterization of antibiotic resistancemarkers and virulence factors.

In contrast to “bottom-up” proteomics, “top-down” proteomics refers tomethods of analysis in which protein samples are introduced intact intoa mass spectrometer, without enzymatic, chemical or other means ofdigestion. Top-down analysis enables the study of the intact protein,allowing identification, primary structure determination andlocalization of post-translational modifications (PTMs) directly at theprotein level. Top-down proteomic analysis typically consists ofintroducing an intact protein into the ionization source of a massspectrometer, fragmenting the protein ions and measuring themass-to-charge ratios and abundances of the various fragmentsso-generated. The resulting fragmentation is many times more complexthan a peptide fragmentation, which may, in the absence of the methodstaught herein, necessitate the use of a mass spectrometer with very highmass accuracy and resolution capability in order to interpret thefragmentation pattern with acceptable certainty. The interpretationgenerally includes comparing the observed fragmentation pattern toeither a protein sequence database that includes compiled experimentalfragmentation results generated from known samples or, alternatively, totheoretically predicted fragmentation patterns. For example, Liu et al.(“Top-Down Protein Identification/Characterization of a Priori UnknownProteins via Ion Trap Collision-Induced Dissociation and Ion/IonReactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer”,Anal. Chem. 2009, 81, 1433-1441) have described top-down proteinidentification and characterization of both modified and unmodifiedunknown proteins with masses up to ≈28 kDa.

An advantage of a top-down analysis over a bottom-up analysis is that aprotein may be identified directly, rather than inferred as is the casewith peptides in a bottom-up analysis. Another advantage is thatalternative forms of a protein, e.g. post-translational modificationsand splice variants, may be identified. However, top-down analysis has adisadvantage when compared to a bottom-up analysis in that many proteinscan be difficult to isolate and purify. Thus, each protein in anincompletely separated mixture can yield, upon mass spectrometricanalysis, multiple ion species, each species corresponding to adifferent respective degree of protonation and a different respectivecharge state, and each such ion species can give rise to multipleisotopic variants. Thus, methods are required for interpreting theresulting highly complex mass spectra.

Ion-ion reactions have found great utility in the field of biologicalmass spectrometry over the last decade, primarily with the use ofelectron transfer dissociation (ETD) to dissociate peptide/proteins anddetermine primary sequence information and characterizepost-translational modifications.

Proton transfer, another type of ion-ion reaction, has also been usedextensively in biological applications. Experimentally, proton transferis accomplished by causing multiply-positively-charged protein ions(i.e., protein cations) from a sample to react with singly-chargedreagent anions so as to reduce the charge state of an individual proteincation and the number of such charge states of the protein cations.These reactions proceed with pseudo-first order reaction kinetics whenthe reagent anions are present in large excess over the protein cationpopulation. The rate of reaction is directly proportional to the squareof charge of the protein cation (or other multiply-charged cation)multiplied by the charge on the reagent anion. The same relationshipalso holds for reactions of the opposite polarity, defined here asreaction between singly-charged reagent cations and a population ofmultiply-charged anions derived from a protein sample. This produces aseries of pseudo-first order consecutive reaction curves as defined bythe starting multiply-charged protein cation population. Although thereactions are highly exothermic (in excess of 100 kcal/mol), protontransfer is an even-electron process performed in the presence of 1mtorr of background gas (i.e. helium) and thus does not fragment thestarting multiply-charged protein cation population. The collision gasserves to remove the excess energy on the microsecond time scale (10⁸collisions per second), thus preventing fragmentation of the resultingproduct ion population.

Proton transfer reactions (PTR) have been used successfully to identifyproteins in mixtures of proteins. This mixture simplification processhas been employed to determine charge state and molecular weights ofhigh mass proteins. PTR has also been utilized for simplifying production spectra derived from the collisional-activation of multiply-chargedprecursor protein ions. Although PTR reduces the overall signal derivedfrom multiply-charged protein ions, this is more than offset by thesignificant gain in signal-to-noise ratio of the resulting PTR productions. The PTR process is 100% efficient leading to only single series ofreaction products, and no side reaction products that require specialinterpretation and data analysis.

Various aspects of the application of PTR to the analysis of peptides,polypeptides and proteins have been described in the followingdocuments: U.S. Pat. No. 7,749,769 B2 in the names of inventors Hunt etal., U.S. Patent Pre-Grant Publication No. 2012/0156707 A1 in the namesof inventors Hartmer et al., U.S. Pre-Grant Publication No. 2012/0205531A1 in the name of inventor Zabrouskov; McLuckey et al., “Ion/IonProton-Transfer Kinetics: Implications for Analysis of Ions Derived fromElectrospray of Protein Mixtures”, Anal. Chem. 1998, 70, 1198-1202;Stephenson et al., “Ion-ion Proton Transfer Reactions of Bio-ionsInvolving Noncovalent Interactions: Holomyoglobin”, J. Am. Soc. MassSpectrom. 1998, 8, 637-644; Stephenson et al., “Ion/Ion Reactions in theGas Phase: Proton Transfer Reactions Involving Multiply-ChargedProteins”, J. Am. Chem. Soc. 1996, 118, 7390-7397; McLuckey et al.,“Ion/Molecule Reactions for Improved Effective Mass Resolution inElectrospray Mass Spectrometry”, Anal. Chem. 1995, 67, 2493-2497;Stephenson et al., “Ion/Ion Proton Transfer Reactions for ProteinMixture Analysis”, Anal. Chem. 1996, 68, 4026-4032; Stephenson et al.,“Ion/Ion Reactions for Oligopeptide Mixture Analysis: Application toMixtures Comprised of 0.5-100 kDa Components”, J. Am. Soc. MassSpectrom. 1998, 9, 585-596; Stephenson et al., “Charge Manipulation forImproved Mass Determination of High-mass Species and Mixture Componentsby Electrospray Mass Spectrometry”, J. Mass Spectrom. 1998, 33, 664-672;Stephenson et al., “Simplification of Product Ion Spectra Derived fromMultiply Charged Parent Ions via Ion/Ion Chemistry”, Anal. Chem., 1998,70, 3533-3544 and Scalf et al., “Charge Reduction Electrospray MassSpectrometry”, Anal. Chem. 2000, 72, 52-60. Various aspects of generalion/ion chemistry have been described in McLuckey et al., “Ion/IonChemistry of High-Mass Multiply Charged Ions”, Mass Spectrom. Rev. 1998,17, 369-407 and U.S. Pat. No. 7,550,718 B2 in the names of inventorsMcLuckey et al. Apparatus for performing PTR and for reducing ion chargestates in mass spectrometers have been described in U.S. Pre-GrantPublication No. 2011/0114835 A1 in the names of inventors Chen et al.,U.S. Pre-Grant Publication No. 2011/0189788 A1 in the names of inventorsBrown et al., U.S. Pat. No. 8,283,626 B2 in the names of inventors Brownet al. and U.S. Pat. No. 7,518,108 B2 in the names of inventors Frey etal. Adaptation of PTR charge reduction techniques to detection andidentification of organisms has been described by McLuckey et al.(“Electrospray/Ion Trap Mass Spectrometry for the Detection andIdentification of Organisms”, Proc. First Joint Services Workshop onBiological Mass Spectrometry, Baltimore, Md., 28-30 Jul. 1997, 127-132).

The product ions produced by the PTR process can be accumulated into oneor into several charge states by the use of a technique known as “ionparking”. Ion parking uses supplementary AC voltages to consolidate thePTR product ions formed from the original variously protonated ions ofany given protein molecule into a particular charge state or states atparticular mass-to-charge (m/z) values during the reaction period. Thistechnique can be used to concentrate the product ion signal into asingle or limited number of charge states (and, consequently, into asingle or a few respective m/z values) for higher sensitivity detectionor further manipulation using collisional-activation, ETD, or other ionmanipulation techniques. Various aspects of ion parking have beendescribed in U.S. Pat. No. 7,064,317 B2 in the name of inventorMcLuckey; U.S. Pat. No. 7,355,169 B2 in the name of inventor McLuckey;U.S. Pat. No. 8,334,503 B2 in the name of inventor McLuckey; U.S. Pat.No. 8,440,962 B2 in the name of inventor Le Blanc; and in the followingdocuments: McLuckey et al., “Ion Parking during Ion/Ion Reactions inElectrodynamic Ion Traps”, Anal. Chem. 2002, 74, 336-346; Reid et al.,“Gas-Phase Concentration, Purification, and Identification of WholeProteins from Complex Mixtures”, J. Am. Chem. Soc. 2002, 124, 7353-7362;He et al., “Dissociation of Multiple Protein Ion Charge States Followinga Single Gas-Phase Purification and Concentration Procedure”, Anal.Chem. 2002, 74, 4653-4661; Xia et al., “Mutual Storage Mode Ion/IonReactions in a Hybrid Linear Ion Trap”, J. Am. Soc. Mass. Spectrom.2005, 16, 71-81; Chrisman et al., “Parallel Ion Parking: ImprovingConversion of Parents to First-Generation Products in Electron TransferDissociation”, Anal. Chem. 2005, 77(10), 3411-3414 and Chrisman et al.,“Parallel Ion Parking of Protein Mixtures”, Anal. Chem. 2006, 78,310-316.

DISCLOSURE OF INVENTION

The present disclosure teaches an application of ion-ion reactionchemistry in which proton transfer reactions are employed to simplifythe mass spectrometric analysis of complex ion populations derived fromelectrospray ionization of samples comprising mixtures of compoundsextracted from microorganisms. The inventors have discovered that bysubjecting a mass-to-charge-restricted subset of such ions to PTR, theresulting population of product ions comprises a much simpler populationof charge states of lower total charge values (where the words “lower”or “reduced”, in this context, refer to lower or reduced in terms ofabsolute value) which can be readily resolved and assigned to specificprotein or peptide ions. Because the PTR product ions represent asmaller subset of multiply-charged species derived from a complexmixture of charge states than the original precursor ions, mass spectralinterpretation is greatly simplified and target analysis using tandemmass spectrometry (MS/MS or MS^(n)) can be performed on a single proteinor other component(s) derived from a microbial extract.

The charge-reduced protein and peptide product ions resulting from agiven proton transfer reaction produce mass-to-charge (m/z) values thatare greater than those of the original m/z values. For a mixture ofprotein ions that have the same m/z value but differing mass and charge,the mixture can be separated on the micro- or millisecond timescale.Further, these multiply-charged protein ions of the same m/z value withdiffering mass and charge can be separated from low m/z value backgroundions derived from small molecules, lipids, solvents, or otherinterferents based on the charge squared dependence of the reaction.Multiply-charged ions are therefore separated in time from thebackground signal thus producing a separated protein mixture at highlyincreased signal-to-noise (s/n) ratio. The inventors have discoveredthat, as a result of these two factors, the spectral signatures of theprotein/peptide or any other analyte product ions may be significantlyseparated from those of most interferent ions. In addition, multiplestages of PTR reactions can be performed to separate protein mixtures onlow resolution instrumentation, such as a linear ion trap massspectrometer, in order to simplify and isolate these proteins and otheranalytes such that target analysis can be performed via MS^(n) analysis.The inventors have further discovered that the advantageous propertiesof simple PTR reactions may be even further amplified by performing “ionparking” procedures in conjunction with PTR reaction, thus enabling ananalyst to at least partially select or control the product-ion chargestate distribution that results from the PTR reaction.

PTR can also be used to improve high mass performance in massspectrometry. In mass spectrometry, an ion may be assigned either aninteger nominal mass or mass-to-charge ratio or an accurate or exactmass or mass-to-charge ratio. Accurate or exact masses or mass-to-chargeratios can be considered as comprising an integer component or value anda decimal component or value. Atomic and molecular masses are measuredin units of daltons (Da) and m/z ratio values are generally given in inunits of daltons per elementary charge, or Da/e or thomson (Th). It isto be noted that, in instances of described numerical values of m/zratios in this document, such ratios are understood to be provided inunits of daltons per elementary charge, or Th. Accurate or exact (i.e.non-integer) masses or m/z ratios can be represented as an integernominal mass or mass-to-charge ratio value or component together with acorresponding decimal component. Thus, as used in this document,accurate mass determination or mass analysis can be considered ascomprising sub-integer accuracy, i.e. accuracy of ±0.5 Da or better and,preferably, 0.1 Da or better.

Alternatively, accurate or exact masses or m/z ratios may be defined interms of parts-per-million (ppm) mass accuracy. For mass spectrometricdeterminations of polypeptides and proteins, an experimental massaccuracy of 50 ppm or better, more preferably 10 ppm or better and,still more preferably 1 ppm or better, is generally required becausesuch molecules and their ions frequently have molecular or ionic weightsof at least 10,000 Da and as much as 100,000 Da. Thus, as used in thisdocument, accurate mass determination or mass analysis can alternativelybe considered as comprising an accuracy of 50 ppm or better, morepreferably 10 ppm or better and, still more preferably, 1 ppm or better.

In addition to improving the signal-to-noise ratios for this type ofanalysis, the inventors have considered that the reduction of charge onprotein ions causes these large ions to refold in the gas phase, as hasbeen described in Zhao et al., “Effects of Ion/Ion Proton TransferReactions on Conformation of Gas-Phase Cytochrome c Ions”, J. Am. Soc.Mass Spec. 2010, 21, 1208-1217. It is believed that this results in amore compact configuration which reduces the collisional cross sectionof the protein ions and, accordingly, increases their stability againstfragmentation by collision with background gas molecules present in themass analyzer chamber. The inventors have discovered that this effectcan be especially beneficial with mass analyzers that employ imagecurrent detection, such as is done in a Fourier-transform ion cyclotronresonance (FT-ICR) mass analyzer or in an Orbitrap™ mass analyzer (atype of electrostatic trap mass analyzer commercially available fromThermo Fisher Scientific of Waltham, Mass. USA). Another potentialreason for improved high mass performance is the large deposition ofenergy into a given protein ion that results from the PTR process. Theenergy deposited as a result of the PTR process exceeds 100 kcal/mol andis then effectively dampened by the presence of collision energy. Thisrapid heating process “boils off” neutral molecules that may be attachedto the protein via ion-dipole, ion-induced dipole, or dipole-induceddipole interactions. Most importantly, the reduction of charge state forhigh mass proteins may significantly improve the transfer of these ionsfrom the relatively high pressure of an ion guide, ion storage or iontrapping device where the PTR process is commonly performed, to alower-pressure region of a mass analyzer, such as an Orbitrap™ massanalyzer. The reduced charge state means that ions are transferred atless kinetic energy thus limiting ion scattering, direct fragmentation,or formation of metastable species. The inventors further consider thatthis latter property is especially significant in enabling high-accuracymass analysis of the PTR product ions in an accurate-massspectrometer—such as the Orbitrap™-type of electrostatic trap massanalyzer—that detects image currents produced by cyclic ionic motionover an extended time range.

The present teachings are especially useful for the analysis andidentification of intact proteins having molecular weight in excess of50 kDa. The inventors have discovered the surprising result that, takentogether, the various advantageous factors noted above can enableaccurate identification of multiple intact proteins or large peptidesfrom even very complex mixtures derived from natural microorganismsamples. Such identifications can enable microorganism identification tothe species, subspecies or even strain level. The target protein orpolypeptide ion single species or multiple species may be chosen so asto be indicative, based on prior knowledge or information, eitherindividually or in combination, of the presence in a sample of aspecific microorganism or cell type, or a specific strain or variant ofa microorganism or cell type, or a given virulence factor or toxin, orof the capacity of a microorganism or cell to resist an antimicrobialcompound or antibiotic drug.

The present invention, in one aspect, offers an alternative totraditional bottom-up proteomics methods, namely top-down analysis ofintact proteins derived from microbial cells via a method which isapplicable to substantially all microorganisms including Gram-positivebacteria, Gram-negative bacteria, mycobacteria, mycoplasma, yeasts,protozoans, filamentous (i.e., microscopic) fungi. The present inventionprovides identification of microorganisms at the genus, species,subspecies, strain pathovar, and serovar level even in samplescontaining mixtures of microorganisms and/or microorganisms analyzeddirectly from pure and/or mixed cultures and from direct samples (e.g.,surface swabs, bodily fluids, etc.). In addition, the approaches taughtherein can be employed for targeted detection of virulence factors,antibiotic resistance and susceptibility markers, or othercharacteristics. The top-down methods of the present teachings aresimple and quick because there is no need for chemical or enzymaticdigestion of a sample and data processing is accomplished in real time.

Methods in accordance with the present teachings may comprise at leastone or more of the following steps: microbial cell disruption,solubilization of proteins, sample clean-up (to desalt, remove insolublecomponents and debris, and/or concentrate), sample infusion or flowinjection, fast partial liquid chromatographic separation, standardchromatographic separation, isoelectric focusing, ionization of proteinsin solution, isolation of a given m/z range of the ions, causing theisolated range of ions to undergo PTR so as to form first-generation PTRproduct ions, optional isolation of an m/z range of the first-generationPTR product ions, optional mass spectrometry in MS or MS/MS mode,optionally causing the isolated range of first-generation PTR productions to undergo a second PTR reaction so as to form second-generationPTR product ions, mass spectrometry in MS or MS/MS mode, and microbialidentification via molecular weight analysis and/or protein sequenceanalysis, or using any statistical classification method. Preferably,but not necessarily, the mass spectrometry steps are performed with ahigh-resolution, high-accuracy mass spectrometer, such as a massspectrometer comprising an Orbitrap™ mass analyzer.

Because a common method using a limited set of chemical reagents isperformed, the methods of the present teachings are suitable for usewithin a completely automated system for sample preparation and massspectrometry. Ideally, these methods may be automated from samplepreparation through results reporting. Results may be automaticallytransferred to a hospital's electronic medical records system where theycan be directly linked to patient treatment strategies, insurance,billing, or used in epidemiological reporting. Such an integrated systemfacilitates epidemiological tracking of an outbreak at the hospital,local, regional, and global levels. For high throughput laboratories,multiple systems can be interfaced to a central computer whichintegrates data from the different instruments prior to reporting. Thesystem can import phenotypic susceptibility data where it can becombined with identification, virulence, antibiotic resistance andtyping information generated by the invention.

Accordingly, in a first aspect, there is disclosed a method foridentifying the presence or absence of a protein/polypeptide or otherbiologically relevant compound within a liquid sample comprising amixture of compounds that includes a plurality of protein compounds or aplurality of polypeptide compounds or pluralities of both protein andpolypeptide or other compounds, wherein the method comprises: (a)introducing a portion or all of the liquid sample into an electrosprayionization source of a mass spectrometer; (b) forming positively-chargedions of the mixture of compounds of the portion of the liquid sample byelectrospray ionization, the positively-charged ions comprising aplurality of ion species; (c) isolating a first subset of the ionspecies comprising a first mass-to-charge (m/z) ratio range thatincludes an m/z ratio of a multiply-protonated molecular species of theanalyte compound; (d) generating a plurality of first-generation production species from the isolated first subset of ion species by causing theisolated first subset of ion species to be reacted, for a predeterminedtime duration, with reagent anions that, upon reaction, extract protonsfrom each of one or more ion species that comprises a protonated speciesof a protein or polypeptide compound; (e) generating a mass spectrum,using a mass analyzer, of either the first-generation product ionspecies or of second-generation product ion species generated from thefirst-generation product ion species; (f) conducting a search of themass spectrum of either the first-generation or the second-generationproduct ion species for a set of one or more m/z ratios that arediagnostic of the protein or polypeptide analyte compound; and (g)making a determination of the presence or absence of the analytecompound within the sample based on a measure of similarity between aset of m/z ratios identified in the mass spectrum and the set of one ormore diagnostic m/z ratios. The measure of similarity may comprise ametric that is calculated based on a determined percentage or proportionof the one or more diagnostic m/z ratios that are found to occur in themeasured set of identified m/z ratios. Alternatively, the presence of ananalyte compound within the sample can be determined by comparing themeasure of similarity between a set of m/z ratios identified in the massspectrum and those contained in a protein, DNA, or carbohydrate baseddatabase; and (h) using the aforementioned information as a way topositively identify any unknown microorganism using spectral libraries,sequence based searching, statistical classification methods includingbut not limited to Bayesian, logistic regression, and decision treeclassifiers. As an alternative to forming positively-charged ions instep (b), negatively-charged analyte ion species may be producedinstead. In such cases, the reagent anions are chosen so as to transferprotons to the analyte ion species, thereby reducing the absolute valuesof their negative charges.

In a second aspect, there is disclosed a method of identifying thepresence or absence of a microorganism type in a sample, comprising: (i)identifying a series of molecular weights whose simultaneous presence inthe sample is diagnostic of the presence of the microorganism type(s) inthe sample (ii) identifying a list of analyte compounds whosesimultaneous presence in the sample is diagnostic of the presence of themicroorganism type(s) in the sample, said list of analyte compoundscomprising protein compounds, polypeptide compounds or both protein andpolypeptide compounds; (iii) extracting, from the sample, a liquidsolution comprising a mixture of sample-derived proteins andpolypeptides; (iv) performing a set of analysis steps for eachrespective analyte compound in the list; and (v) identifying thepresence of the microorganism(s) type within the sample if the presenceof microorganism specific analyte compounds of the list of analytecompounds is identified within the liquid solution. The analysis stepsthat are performed for each respective analyte in the list comprise: (a)introducing a portion of the liquid solution into an electrosprayionization source of a mass spectrometer; (b) forming positively-chargedions of the mixture of compounds of the portion of the liquid solutionby electrospray ionization, the positively-charged ions comprising aplurality of ion species; (c) isolating a first subset of the ionspecies comprising a first mass-to-charge (m/z) ratio range thatincludes an m/z ratio of either a random or particular predeterminedmultiply-protonated molecular species of the respective analytecompound; (d) generating a plurality of first-generation product ionspecies from the isolated first subset of ion species by causing theisolated first subset of ion species to be reacted, for a predeterminedtime duration, with reagent anions that spontaneously extract protonsfrom each of one or more ion species that comprises a protonated speciesof a protein or polypeptide compound; (e) generating a mass spectrum,using a mass analyzer, of either the first-generation product ionspecies or of second-generation product ion species generated from thefirst-generation product ion species; (f) conducting a search of themass spectrum of either the first-generation or the second-generationproduct ion species for a set of one or more m/z ratios that arediagnostic of the respective analyte compound; and (g) identifying thepresence of the respective analyte compound within the liquid solutionbased on a measure of similarity between a set of m/z ratios identifiedin the mass spectrum and the set of one or more diagnostic m/z ratios.The measure of similarity may comprise a metric that is calculated basedon a determined percentage or proportion of the one or more diagnosticm/z ratios that are found to occur in the measured set of identified m/zratios. The diagnostic m/z ratios can be derived from a spectral libraryor sequence database. If the m/z ratio that is isolated in step (c) isof a random multiply-protonated molecular species, then the searchconducted in step (f) is a sequence-based search. Otherwise, if the m/zratio that is isolated in step (c) is of a particular predeterminedmultiply-protonated molecular species, then a spectral library search isconducted in step (f). In addition to using the aforementionedinformation as a way to positively identify an unknown microorganismusing spectral libraries or sequence based searches, statisticalclassification methods including but not limited to Bayesian, logisticregression, and decision tree classifiers can be utilized for microbialcharacterization and identification. As an alternative to formingpositively-charged ions in step (b), negatively-charged analyte ionspecies may be produced instead. In such cases, the reagent anions orcations are chosen so as to transfer protons to the analyte anionspecies, thereby reducing the absolute values of their negative charges.Control of the PTR experimental processes described herein can beperformed manually or automatically in real-time using real-timespectral deconvolution.

The term “real-time spectral deconvolution” in the above refers tospectral deconvolution of mass spectral data that is performedconcurrently with the mass spectral experiment or analytical run thatgenerates (or that has generated) that mass spectral data. For example,mass spectral data acquired by mass analysis of analytes that elute at afirst retention chromatographic retention time during a gradient elutionmay be deconvoluted, so as to identify the analytes, simultaneously withthe continued collection of additional mass spectral data of additionalanalytes that elute at a second, later retention time during the samegradient elution. Likewise, deconvolution of the additional massspectral data, so as to identify the additional analytes, may beperformed simultaneously with the continued collection of mass spectraldata of analytes that elute at a third elution time during the samegradient elution. The real-time spectral deconvolution may befacilitated by the use of a fast computer, such as a computer thatemploys parallel processing or a graphics processing unit (GPU) toperform the necessary calculations. Alternatively or additionally, thereal-time spectral deconvolution may be facilitated by the use of acomputationally efficient or optimized algorithm, such as an algorithmthat is written at least partially in assembly language or that makesextensive use of in-cache look-up-tables.

More generally, the term “real-time” may be understood as meaning, whenused in reference to an event or activity associated with a dataacquisition process, that the event or activity occurs while some aspector sub-process of that data acquisition process is ongoing. The dataacquisition process itself may include one of more the followingindividual sub-processes: sample purification (e.g., solid phaseextraction, size-exclusion chromatography); sample separation (e.g.,chromatography); sample transfer into a mass spectrometer (e.g.,infusion or inletting of eluate from a chromatograph); sample ionizationin an ion source to as to generate first-generation ions; selection andisolation of ions for further manipulation; causing fragmentation ofsample-derived ions or reaction of sample-derived ions with reagent ionsso as to generate a first-generation of product ions; optional selectionand isolation of product ions; optional further fragmentation of productions or further reaction of product ions; transfer of ions(first-generation ions or first-generation or subsequent-generationproduct ions) to a mass analyzer, detection and measurement of ionmass-to-charge ratios by a detector of the mass analyzer; and transferof data derived from the detection and measurement to a digitalprocessor for storage, mathematical analysis, etc. The events oractivities that may occur in “real-time”, so defined, may include, butare not necessarily limited to: determination or identification of thepresence of an analyte in a sample; identification or determination ofthe presence of a microorganism in a sample and providing a notificationto a user of the identification or determination of the presence of ananalyte or microorganism in a sample.

The above-described and various other features and advantages of thepresent teachings will become more fully apparent from the followingdescription and appended claims, or may be learned by the practice ofthe invention as set forth hereinafter.

BRIEF DESCRIPTION OF DRAWINGS

To further clarify the above and other advantages and features of thepresent disclosure, a more particular description of the disclosure willbe rendered by reference to specific embodiments thereof, which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the disclosure and aretherefore not to be considered limiting of its scope. The disclosurewill be described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 is a block diagram schematically illustrating a system for rapidextraction and analysis of soluble proteins from at least onemicroorganism for identifying the at least one microorganism;

FIG. 2 is a schematic representation of an exemplary mass spectrometersuitable for employment in conjunction with methods according to thepresent teachings, the mass spectrometer comprising a hybrid systemcomprising a quadrupole mass filter, a dual-pressure quadrupole ion trapmass analyzer and an electrostatic trap mass analyzer;

FIG. 3A is a flow diagram of a first method in accordance with thepresent teachings;

FIG. 3B is a flow diagram of an alternative method in accordance withthe present teachings;

FIG. 3C is a flow diagram of another alternative method in accordancewith the present teachings;

FIG. 3D and FIG. 3E illustrate a flow diagram of yet another alternativemethod in accordance with the present teachings;

FIG. 3F is a flow diagram of still yet another alternative method inaccordance with the present teachings;

FIG. 4A is an ESI mass spectrum via direct infusion of a typical E. coliextract;

FIG. 4B is a PTR product-ion mass spectrum generated by isolating ionsof the E. coli extract of FIG. 4A within a 2 Th mass window centered atm/z=750 Th and reacting the isolated ions with PTR reagent anions;

FIG. 5A is a mass spectrum of first-generation PTR product ionsgenerated by isolating ions of an E. coli extract within a mass windowof width 5 Th centered at 1200 Th and reacting the isolated ions withPTR reagent anions;

FIG. 5B is a mass spectrum of second-generation PTR product ionsgenerated by isolating ions of the first-generation PTR product ions ofFIG. 5A within a mass window of width 5 Th centered at 1320 Th andreacting the isolated first-generation product ions with PTR reagentanions a second time;

FIG. 6A is a mass spectrum of PTR product ions generated by isolatingions of an E. coli extract within a mass window of width 5 Th centeredat 640 Th and reacting the isolated ions with PTR reagent anions;

FIG. 6B is a mass spectrum of an isolated PTR product ion speciesselected from the product ion assemblage of FIG. 6A and having an m/zratio of 833 Th;

FIG. 6C is a mass spectrum of second-generation product ions generatedby collision-induced dissociation (CID) of the isolated PTR product ionspecies of FIG. 6B;

FIG. 6D is a mass spectrum of an isolated PTR product ion speciesselected from the product ion assemblage of FIG. 6A and having an m/zratio of 926 Th;

FIG. 6E is a mass spectrum of second-generation product ions generatedby collision-induced dissociation of the isolated PTR product ionspecies of FIG. 6D;

FIG. 6F is a mass spectrum of an isolated PTR product ion speciesselected from the product ion assemblage of FIG. 6A and having an m/zratio of 917 Th;

FIG. 6G is a mass spectrum of second-generation product ions generatedby collision-induced dissociation of the isolated PTR product ionspecies of FIG. 6F;

FIG. 7A is a schematic depiction of a method, in accordance with thepresent teachings, of improved-efficiency PTR conversion of ions of aselected analyte to an assemblage of PTR product ions by simultaneousisolation and reaction of multiple m/z ranges of electrospray-producedfirst-generation precursor ions;

FIG. 7B is a schematic diagram of isolation of a first randomly-chosenrange of electrospray-produced first-generation precursor ions for PTRreaction, as may be employed in an initial step of a method ofimproved-efficiency PTR conversion of ions;

FIG. 7C is a schematic depiction of recognition of two charge-statesequences of PTR product ions corresponding to different analytemolecules, as may be employed as an intermediate step of a method ofimproved-efficiency PTR conversion of ions;

FIG. 8 is a flow diagram of a method, in accordance with the presentteachings, of improved-efficiency PTR conversion of ions of a selectedanalyte to an assemblage of PTR product ions;

FIG. 9A is a full scan mass spectrum of first-generation ions generatedfrom eluate at a retention time of 10 min. and 30 s. during the courseof a ten-minute gradient reverse-phase liquid chromatography separationof an E. coli extract;

FIG. 9B is a PTR product ion spectrum generated by reacting sulfurhexafluoride for 10 ms with an isolated population of ions of the sampleof FIG. 9A within a 10 Th wide isolation window centered at 750 Th;

FIG. 10A is a full scan mass spectrum of first-generation ions generatedfrom eluate at a retention time of 42 min. and 30 s. during the courseof a sixty-minute gradient reverse-phase liquid chromatographyseparation of an E. coli extract;

FIG. 10B is a PTR product ion spectrum generated by reacting sulfurhexafluoride for 10 ms with an isolated population of ions of the sampleof FIG. 10A within a 10 Th wide isolation window centered at 750 Th;

FIG. 11A is a full scan mass spectrum of first-generation ions generatedfrom eluate at a retention time of 18 min. and 9 s. during the course ofa thirty-minute gradient reverse-phase liquid chromatography separation;

FIG. 11B is a PTR product ion spectrum generated by reaction of PTRreagent ions with an isolated population of ions of the sample of FIG.11A within a 10 Th wide isolation window centered at 750 Th;

FIG. 11C is a full scan mass spectrum of first-generation ions generatedfrom eluate at a retention time of 22 min. and 27 s. during the courseof the same thirty-minute gradient reverse-phase liquid chromatographyseparation of which the earlier elution results are plotted in FIG. 11A;and

FIG. 11D is a PTR product ion spectrum generated by reaction of PTRreagent ions with an isolated population of ions of the sample of FIG.11C within a 10 Th wide isolation window centered at 750 Th.

MODES FOR CARRYING OUT THE INVENTION

The following description is presented to enable any person skilled inthe art to make and use the invention, and is provided in the context ofa particular application and its requirements. Various modifications tothe described embodiments will be readily apparent to those skilled inthe art and the generic principles herein may be applied to otherembodiments. Thus, the present invention is not intended to be limitedto the embodiments and examples shown but is to be accorded the widestpossible scope in accordance with the claims. The particular featuresand advantages of the invention will become more apparent with referenceto the appended FIGS. 1-11 taken in conjunction with the followingdescription.

Referring now to FIG. 1, a system 100 for extraction of proteins fromone or more microorganisms, detection of the proteins, andidentification of the one or more microorganisms is schematicallyillustrated. The system 100 includes a sample handling device 115, asample 110 that is accessible by the sample handling device 115, andsources of reagents, buffers, and the like 120, these sources beingfluidly coupled to the sample handling device 115 by various tubing orother transfer lines. The system 100 further includes a first and,optionally, a second sample-purification device 135 (such as a solidphase extraction cartridge) configured for cleaning up samples (e.g.,desalting, removing contaminants, concentrating proteins) and anoptional chromatography column 140 that may be configured for at leastpartially purifying a sample 110 by liquid chromatography prior tomass-spec analysis. At least one sample-purification device 135 cancomprise an in-line size exclusion chromatography column that can beused to not only remove salts but small molecules and lipids as well.The sample 110, the first and optional second sample-purificationdevices 135, and the optional chromatography column 140 are in fluidcommunication with a fluid handling pump 130, the various reagents,buffers and other fluids 120, and a mass spectrometer 150.

The sample handling device 115 is capable of preparing a range of sampletypes containing one or more microbes and delivering a soluble proteinfraction extracted from the microbes to the mass spectrometer 150 foranalysis. A sample 110 may be of any type suspected to contain one ormore microorganisms including, without limitation, isolated coloniesfrom a culture plate, cells from liquid growth medium, blood, bloodculture, saliva, urine, stool, sputum, wound and body site swabs, soil,food, beverage, water, air, and environmental surface swabs.

The sample handling device 115 may include one or more of a celldisruption means, a robotic liquid handling means, a centrifuge,filtration means, an incubator, mixing means, a vacuum pump, a fluidpump, and reagents 120 that can be used for disruption of microbes andisolation of a soluble protein fraction. Disruption of bacterial,fungal, mycoplasma cells, viruses, and the like may be achieved bymechanical, chemical, enzymatic and other means as are commonly known inthe art. Mechanical approaches include bead beating, use of pressurelike French press and the like, sonication or other methods known in theart. Chemical methods include exposure to chaotropes such as urea,thiourea, or guanidine HCL to lyse the microbial cells and solubilizetheir contents. Alternatively, organic acid/solvents mixtures may beutilized to disrupt cells. Enzymatic methods include using lysozyme,lysostaphin or other lytic enzymes to form “holes” in the bacterial cellwalls that allow the contents to leak out into the surrounding solution.

As illustrated in FIG. 1, the system 100 further includes an optionalcontrol unit 160 that can be linked to various components of the system100 through linkages 170 a-170 d. For example, the control unit 160 canbe linked to the sample 110 to control sample application, the reagents120 to control the application of various reagents, the pump 130 tocontrol fluid handling, flow rates, etc., to the sample handling device115 to control sample preparation, and to the mass spectrometer 150 tocontrol mass spectrometry parameters. In the illustrated embodiment, thecontrol unit 160 can also serve as a data processing unit to, forexample, process data from the mass spectrometer 150 or to forward thedata to server(s) for processing and storage (the server is not shown inFIG. 1). Control unit 160 can also determine molecular weights andcharge states of any generation of PTR product ions for MS/MS, MS^(n),or molecular weight determination in real time. The Control Unit 160 canalso be used to automatically forward the results to health careprofessionals.

In some embodiments, the system 100 is designed to be used by aclinician or a general laboratory technician who is not necessarilyexpert in all aspects of sample preparation, LC-MS operations, LC-MSmethods development, and the like. As such, the control unit 160 can bedesigned to encapsulate the data system environment by providing a userwith a simplified application interface that can be used to initiate andmonitor essentially all aspects of assaying a sample 110 withoutrequiring the user to interact with the overall hardware and controlsystems of the system 100. The control unit 160 is therefore configuredto provide a degree of separation between the user and the underlyingservices that control devices, data files and algorithms for translatingdata to a user readable form. That is, the control unit 160 eliminatesthe need for the user to be aware of or in control of hardware foranalyzing clinical samples and provides a simplified interface to sendand receive information from the mass spectrometer.

The control unit 160 may be configured to internally monitor each sampleanalysis request and is capable of tracking the analysis request fromstart to finish through the system 100. Once data for a sample 110 isbeing acquired or has been acquired by the system 100, the control unit160 may be configured to automatically start post processing the databased on the type of assay selected by the user. Most importantly, thecontrol unit 160 can be configured to process data in real time duringthe acquisition process. Here results are returned to the user inreal-time that include microbial identification, virulence andresistance characterization, strain matching information, and data onantibiotic susceptibility testing. Moreover, the control unit 160 can beconfigured to automatically select post-processing parameters based onthe type of assay selected by the user, further reducing the need forthe user to interact with the system once the assay has been selectedand started for analysis. The control unit 160 can be designed as alayer that fits between the system 100 and the user to reduce thecomplexity needed to set up sample assays for acquisition. The controlsystem 160 can also be configured to return only the most relevant datato the user to avoid overwhelming the user with extraneous information.

In one embodiment, the system 100 can further include a sample detectiondevice (not pictured) operably coupled to or integrated with the samplehandling device 115. The sample detection device can work with thesample handling device 115 or independently of the sample handlingdevice 115 perform at least one of the following functions: i. identifysamples entering the system; ii. identify assay types for the samplesentering the system; iii. select an assay protocol based on theanticipated assay type and/or analyte of interest; iv. direct the samplehandling device and/or the control system to initiate analysis of theanalyte of interest in the sample; v. direct the control system toselect one or more reagents based upon the assay protocol selected forthe type of assay and/or analyte of interest; vi. direct the controlsystem to select a liquid chromatography mobile phase condition basedupon the assay protocol selected for the type of assay and/or analyte ofinterest and cause the liquid chromatography system to perform the assayand/or purify the analyte of interest; vii. direct the control system toselect a mass spectrometer setting based upon the assay protocolselected for the assay type and/or analyte of interest and cause themass spectrometer to create mass spectral data associated with theselected assay type and/or analyte of interest; and viii. direct thecontrol system to analyze the mass spectral data associated with theselected assay type and/or analyte of interest to identify the presenceand/or concentration of the analyte of interest.

The sample, or the processed sample, may be cleaned up and or purifiedprior to analysis by mass spectrometry. Such purification, or sampleclean-up, may refer to a procedure that removes salts or lipids from thecrude cell extract, or to a procedure that enriches one or more analytesof interest relative to one or more other components of the sample. Italso may refer to sample processing and clean-up in a separatelaboratory that has biosafety level-three facilities for handlingmycobacteria or filamentous fungi. In this embodiment samples aretransferred to the system and can be analyzed as described previously.In one embodiment, such purification, or sample clean-up, may beaccomplished by a solid phase extraction device, in-line size exclusionchromatography and/or the optional chromatography column 140.

In one embodiment, the first and/or second sample-purification device135 may include a solid phase extraction (SPE) cartridge. In someembodiments, the SPE cartridge may be in line directly with the highresolution/high mass accuracy mass spectrometer 150. In one embodiment,the SPE cartridge may be a polypropylene tip with a small volume ofsilica or other sorbent containing bonded C₄, C₈ or C₁₈ or otherfunctional groups immobilized in the cartridge, for example, a StageTip™cartridge (Thermo Fisher Scientific). In alternative embodiments,polymeric sorbents or chelating agents may be used. The bed volume maybe as small as 1 μL or less but greater volumes may also be used. Theapparatus and method are well suited to the complex samples derived fromthe microbial cells because each SPE cartridge is used only once,minimizing carryover problems from one sample to another.

In one embodiment, a sample-purification device 135 may be an in-linesize-exclusion chromatography column designed to remove salts, smallmolecules, and lipids from the sample 110. The approach can be used toseparate medium and large molecular weight proteins as well. Phases areselected to be compatible with partial (i.e., less than 100 percent)organic solutions and organic acids. Phases can accommodate protein sizedistributions that differ in molecular weight from 10³ to 10⁸ Da. Flowrates are adjusted in real time to effect separation of intact proteinsfrom small molecules with separation flow rates typically much less thanthe higher flow rates used to remove small molecules, lipids, and saltsfrom the system. In this embodiment, a sample-purification device 135may also be heated to facilitate faster diffusion rates for intactproteins, thus significantly shortening run times. The flow of mobilephase through a sample-purification device 135 may also be divertedduring a portion of the clean-up process to remove certain impuritiesfrom the flow stream and prevent them from entering the massspectrometer 150.

In one embodiment, the optional chromatography column 140 may include acolumn configured for at least partial chromatographic separation of theproteins in the sample. The stationary phase in the chromatographycolumn may be porous or non-porous silica or agarose particles, or amonolithic material polymerized or otherwise formed inside the column.The stationary phase may be coated with an appropriate material such asC₁₈, C₈, C₄ or another suitable derivative, or contain cation exchangeror other material, or the combination of the above to facilitate theseparation of the proteins, and such material may be chemically bondedto the particles or monolith inside the column. Particle sizes typicallyrange from about 1.5 μm to 30 μm. Pore sizes can range from 50 to 300angstroms. Inside diameters of columns typically range from about 50 μmto 2.1 mm, and column length from about 0.5 cm to 25 cm, or other. Themobile phase or eluent may be a pure solvent, or a mixture of two ormore solvents, and may contain added salts, acids and/or other chemicalmodifiers. The proteins are separated on the column based on one or morephysiochemical properties, including size, net charge, hydrophobicity,affinity, or other physiochemical properties. Chromatographic separationmethods include one or more of ion exchange, size exclusion, HILIC,hydrophobic interaction, affinity, normal-phase, or reverse-phasechromatography.

Additional methods of purifying the samples may include, withoutlimitation, liquid chromatography, HPLC, UHPLC, precipitation,solid-phase extraction, liquid-liquid extraction, dialysis, affinitycapture, electrophoresis, filtration, ultra-filtration or other suitablemethods known in the art for purification.

Various methods have been described involving the use of HPLC for sampleclean-up prior to mass spectrometry analysis. One of skill in the artcan select HPLC instruments and columns that are suitable for use in theinvention. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties inspace and time. The medium may include very small particles, which mayhave a bonded surface that interacts with the various chemical moietiesto facilitate separation of the analytes of interest. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedsurface. Alkyl bonded surfaces may include C₄, C₈, or C₁₈ bonded alkylgroups. In addition, monolithic and other phases known in the state ofthe art may be used as well. The chromatographic column includes aninlet port for receiving a sample and an outlet port for discharging aneffluent that includes the fractionated sample. For example, a testsample may be applied to the column at the inlet port, eluted with asolvent or solvent mixture, and discharged at the outlet port. Inanother example, more than one column may be used sequentially or as atwo-dimensional (2D) chromatography system wherein a test sample may beapplied to a first column at the inlet port, eluted with a solvent orsolvent mixture onto a second column, and eluted with a solvent orsolvent mixture from the second column to the outlet port. Differentsolvent modes may be selected for eluting the analytes. For example,liquid chromatography may be performed using a gradient mode, anisocratic mode, or a polytyptic (i.e. mixed) mode.

FIG. 2 is a schematic depiction of an exemplary mass spectrometer 150 awhich may be employed as the mass spectrometer 150 of FIG. 1. The massspectrometer illustrated in FIG. 2 is a hybrid mass spectrometer,comprising more than one type of mass analyzer. Specifically, the massspectrometer 150 a includes an ion trap mass analyzer 216 as well as anOrbitrap™ analyzer, which is a type of electrostatic trap mass analyzer.Since, as will be described below, various analysis methods inaccordance with the present teachings employ multiple mass analysis dataacquisitions, a hybrid mass spectrometer system can be advantageouslyemployed to improve duty cycles by using two or more analyzerssimultaneously. The Orbitrap™ mass analyzer 212 employs image chargedetection, in which ions are detected indirectly by detection of animage current induced on an electrode by the motion of ions within anion trap.

In operation of the mass spectrometer 150 a, an electrospay ion source201 provides ions of a sample to be analyzed to an aperture of a skimmer202, at which the ions enter into a first vacuum chamber. After entry,the ions are captured and focused into a tight beam by a stacked-ringion guide 204. A first ion optical transfer component 203 a transfersthe beam into downstream high-vacuum regions of the mass spectrometer.Most remaining neutral molecules and undesirable high-velocity ionclusters, such as solvated ions, are separated from the ion beam by acurved beam guide 206. The neutral molecules and ion clusters follow astraight-line path whereas the ions of interest are caused to bendaround a ninety-degree turn by a drag field, thereby producing theseparation.

A quadrupole mass filter 208 of the mass spectrometer 150 a is used inits conventional sense as a tunable mass filter so as to pass ions onlywithin a selected narrow m/z range. A subsequent ion optical transfercomponent 203 b delivers the filtered ions to a curved quadrupole iontrap (“C-trap”) component 210. The C-trap 210 is able to transfer ionsalong a pathway between the quadrupole mass filter 208 and the ion trapmass analyzer 216. The C-trap 210 also has the capability to temporarilycollect and store a population of ions and then deliver the ions, as apulse or packet, into the Orbitrap™ mass analyzer 212. The transfer ofpackets of ions is controlled by the application of electrical potentialdifferences between the C-trap 210 and a set of injection electrodes 211disposed between the C-trap 210 and the Orbitrap™ mass analyzer 212. Thecurvature of the C-trap is designed such that the population of ions isspatially focused so as to match the angular acceptance of an entranceaperture of the Orbitrap™ mass analyzer 212.

Multipole ion guide 214 and optical transfer component 203 b serve toguide ions between the C-trap 210 and the ion trap mass analyzer 216.The multipole ion guide 214 provides temporary ion storage capabilitysuch that ions produced in a first processing step of an analysis methodcan be later retrieved for processing in a subsequent step. Themultipole ion guide 214 can also serve as a fragmentation cell. Variousgate electrodes along the pathway between the C-trap 210 and the iontrap mass analyzer 216 are controllable such that ions may betransferred in either direction, depending upon the sequence of ionprocessing steps required in any particular analysis method.

The ion trap mass analyzer 216 is a dual-pressure linear ion trap (i.e.,a two-dimensional trap) comprising a high-pressure linear trap cell 217a and a low-pressure linear trap cell 217 b, the two cells beingpositioned adjacent to one another separated by a plate lens having asmall aperture that permits ion transfer between the two cells and thatpresents a pumping restriction and allows different pressures to bemaintained in the two traps. The environment of the high-pressure cell217 a favors ion cooling, ion fragmentation by either collision-induceddissociation or electron transfer dissociation or ion-ion reactions suchas proton-transfer reactions. The environment of the low-pressure cell217 b favors analytical scanning with high resolving power and massaccuracy. The low-pressure cell includes a dual-dynode ion detector 215.

The use of either a step of electron transfer dissociation or protontransfer reaction within a mass analysis method requires the capabilityof causing controlled ion-ion reaction within a mass spectrometer.Ion-ion reactions, in turn, require the capabilities of generatingreagent ions and of causing the reagent ions to mix with sample ions.The mass spectrometer 150 a, as depicted in FIG. 2, illustrates twoalternative reagent-ion sources, a first reagent-ion source 299 adisposed between the stacked-ring ion guide 204 and the curved beamguide 206 and a second reagent-ion source 299 b disposed at the oppositeend of the instrument, adjacent to the low-pressure cell 217 b of thelinear ion trap mass analyzer 216. Generally, any particular system willonly include one reagent ion source at most. However, two differentreagent ion sources are depicted and discussed here for illustrativepurposes. Although the following discussion is directed to reagent ionsources for PTR, similar discussion may apply to ETD reagent ionsources.

A first possible reagent ion source 299 a may be located between thestacked ring ion guide 204 and the curved beam guide 206. The reagention source 299 a comprises a glow discharge cell comprising a pair ofelectrodes (anode and cathode) that are exposed to a reagent gas conduit298 a that delivers the reagent gas from a reagent liquid (or solid)reservoir 297 a having a heater that volatilizes the reagent compound.When a high voltage is applied across the electrodes, glow discharge isinitiated which ionizes the reagent flowing between the electrodes.Reagent anions from the glow discharge source are introduced into theion optics path ahead of the quadrupole mass filter 208 within whichthey may be m/z selected. The reagent ions may then be accumulated inthe multipole ion guide 214, and subsequently transferred into the highpressure cell 217 b of the dual-pressure linear ion trap 216 withinwhich they are made available for the PTR reaction. The reactionproducts may be directly transferred to the low pressure cell 217 a orto the Orbitrap™ mass analyzer 212 for m/z analysis.

A possible alternative reagent ion source 299 a may be located adjacentto the low pressure linear trap cell 217 b where it may comprise anadditional high-vacuum chamber 292 from which reagent ions may bedirected into the high pressure cell 217 b through an aperture inbetween chamber 292 and the high-pressure cell. In operation, gaseousreagent compound is supplied from a reagent liquid (or solid) reservoir297 b having a heater that volatilizes the reagent compound and isdirected through a reagent gas conduit 298 b that delivers the reagentgas into a partially confined ion generation volume 296. In operation,thermionic electrons supplied from an electrically heated filament 294are directed into the ion generation volume 296 with a certainpre-determined energy by application of an electrical potential betweenthe filament 294 and an accelerator electrode (not shown). The suppliedenergetic electrons cause ionization of the reagent gas so as togenerate reagent ions. The reagent ions may then be guided into the highpressure cell 217 b by ion optical transfer component 203 a under theoperation of gate electrodes (not shown).

Exemplary methods in accordance with the present teachings areschematically illustrated in the flow diagrams shown in FIGS. 3A-3F.FIG. 3A schematically illustrates a first such exemplary method, method300, for monitoring for the presence of and, optionally, quantifying,certain specific targeted analyte proteins or peptides in a sample ofmicroorganisms. The initial steps 302, 304 and 306 of the method 300 arethe steps of microorganism disruption (e.g., lysis) and extraction,solid-phase clean-up, or size-exclusion chromatography andchromatographic separation, respectively, as described above. In someexperimental situations, the extracted sample may be directly infusedinto a mass spectrometer in the subsequent sample introduction step 308;thus, the steps 304 and 306 are shown by dashed lines as being optional.Samples may also be prepared using offline approaches includingdialysis, or other techniques known in the state of the art. However, inmany other experimental situations, the steps 304 and 306 are useful soas to at least partially purify the sample prior to mass-spectralanalysis.

When an analysis must be completed according to time constraints, as insome clinical applications, the required time for the analysis may beshortened by employing either a SPE step 304, a time-compressedchromatography step as described in U.S. Pat. No. 5,175,430 to inventorEnke, or the method of “Fast Partial Chromatographic Separation” (FPCS)in the chromatography step 306 as described in international (PCT)patent application publication WO 2013/166169 A1. Generally, inperforming FPCS, a crude extract of microbial cells containing a complexmixture of various organic and inorganic analytes (small organicmolecules, proteins and their naturally occurring fragments, lipids,nucleic acids, polysaccharides, lipoproteins, etc.) is loaded on achromatographic column and subjected to chromatography. However, insteadof allowing a gradient to elute each analyte separately (ideally, oneanalyte per chromatographic peak), the gradient is intentionallyaccelerated to the extent that substantially no chromatographic peaksobtained for example approximately eight minutes or less, and preferablyfive minutes or less instead of a much longer run time that would berequired to obtain a baseline separation. In the FPCS separation, manyanalytes are intentionally co-eluted from the column at any given timeaccording to their properties and the type of chromatography (reversephase, HILIC, etc.) used. Partial or incomplete separation may be alsoaccomplished by other methods known to one skilled in the art, includingbut not limited to the use of mobile phase solvents and/or modifiersthat reduce retention of compounds on the column, selection ofstationary phase media that reduce retention of compounds on the column(including particle size, pore size, etc.), operation of thechromatographic system at higher flow rate, operation of thechromatographic system at an elevated temperature, or selection of adifferent chromatographic separation mode (i.e., reversed-phase, sizeexclusion, etc.). The FPCS technique yields few or, possibly, noresolved chromatographic peaks across the whole gradient. Thus,substantially the only relevant information derived from a chromatogramis the time of elution from the column. Each mass spectrum that isrecorded represents a “subset” of co-eluting analytes that is thenionized, separated in the mass analyzer and detected.

In step 308 (FIG. 3A), the sample is introduced into a massspectrometer. The sample may be provided as the eluate material thatemerges from an SPE cartridge, a chromatography apparatus or,alternatively, by direct infusion of the eluate solution. Upon beingprovided to the mass spectrometer, the sample compounds are ionized(step 308) by an electrospray ionization source of the massspectrometer. These electrospray-generated ions are herein referred toas “first-generation” ions. At this juncture, a full or segmented MS¹scan may optionally be performed (step 309) in order to identify theprotein-rich regions in m/z space. (Note that in this document, the term“scan” may be taken to generally refer to a mass spectrum when used as anoun or, alternatively, to the acquisition of a mass spectrum, when usedas a verb). In a preferred embodiment, the MS¹ scan can be obtained overthe full mass range of the mass spectrometer instrument in order to beable to subsequently choose, in data-dependent or independent fashion,an information-rich portion of the spectrum for isolation (step 310).However, in the case of a targeted analysis, the MS¹ scan may beunnecessary and execution of the method 300 may proceed directly to step310, in which a subset of the ions is then isolated for further reactionand analysis. When targeted analysis is employed, the isolationperformed in step 310 may be such that ions within a certainpre-determined m/z range or possibly multiple pre-determined m/z rangesare retained for the subsequent reaction and analysis whereas ionsoutside the pre-determined m/z range or ranges are discarded. Thepre-determined m/z range or ranges are chosen so as to correspond topreferably known m/z ratios of targeted analyte proteins or peptideswhose presence or quantity is detected or monitored in the execution ofthe method.

Generally, the isolation of step 310 may be performed, in known fashion,by introducing the ions from the ion source into an ion trap—such as athree-dimensional ion trap, a curved ion trap (sometimes referred to asa “C-Trap”) a single segment linear ion trap, multiple segmented linearion trap, multipole ion guide or quadrupole mass filter—and thenresonantly ejecting the ions whose m/z ratios are outside of the desiredrange by applying a supplemental AC voltage across pairs of electrodesof the ion trap or applying the appropriate RF/DC voltage ratios toisolate the ion population of interest. In some embodiments, thefrequency of the supplemental voltage may be swept through variousfrequencies such that the ions are ejected in sequence according totheir m/z ratios. In such cases, the ions may be detected as they areejected so as to generate a mass spectrum of the original set of ions.However, since a mass spectrum may not be required at this stage, thesupplemental AC voltage may be alternatively applied as a combination ofsuperimposed frequencies that are chosen so as to cause essentiallysimultaneous ejection of the ions whose whose m/z ratios are outside ofthe desired range. In some embodiments, the combination of superimposedfrequencies may be provided with multiple segments of missingfrequencies (i.e., “notches”) such that ions comprising two or morenon-contiguous m/z ratio ranges are simultaneously isolated within thetrap. Each one of the non-contiguous m/z ratio ranges may correspond toa preferably known m/z ratio of a respective unique targeted analyteprotein or peptide. The applied RF/DC voltage ratios of a quadrupolemass filter may also be used to isolate the defined or targeted massranges of interest. Particular m/z ranges of the first-generation ionsare selected by a single or series of fixed RF/DC voltage ratios inorder to select the appropriate mass isolation windows. The instrumentalconfiguration employed in this case may be a hybrid mass spectrometerinstrument comprising a quadrupole, a C-trap, an Orbitrap™ massanalyzer, and a high energy collision cell (HCD) where the isolated ionpopulation can be stored in either the C-trap or HCD cell for PTRexperiments. The isolated population or populations of thefirst-generation ions are herein referred to as “precursor” ions,because these ions will be subjected to subsequent ion-ion reactions orto fragmentation.

In a preferred embodiment, the isolation of the precursor ion populationmay be performed in a first segment of a segmented linear ion trap.After isolation of the desired ion population, the multiply-chargedprotein ion population may be advantageously moved to another segment ofthe linear ion trap. These steps can be repeated multiple times forisolated defined ranges of precursor ions prior to the PTR process.

Next, anions are generated using either a rhenium-based filament withchemical ionization or glow discharge ionization source from a suitablehigh electron affinity based gaseous reagent. Chemical ionization can beperformed using nitrogen, methane, isobutane, or other known gases inthe state of the art. The anion reagent may be a gas at room temperatureor may be a liquid with sufficient vapor pressure to produce an excessof anions which will drive the PTR process under pseudo-first orderreaction conditions. The anions are then transferred from the sourceregion to the segmented linear trap whereby the specific anion reagentis mass isolated using supplemental AC voltages as described above. Theanion source can be in-line with the electrospray source or mounted onthe opposite end of the segmented linear ion trap. Alternatively, aquadrupole mass filter can perform the anion isolation as well with thesubsequent PTR process occurring in the C-trap or HCD cell of theinstrument.

In step 312 of the method 300 (FIG. 3A), the ions which weremass-isolated in step 310 (i.e., “precursor” ions) are subjected to aproton transfer reaction in which a reagent anion species is reacted fora specified time period with the sample precursor ions in the ion trapso as to extract protons from the precursor cations. In one embodiment,the multiply-charged precursor ion population and the singly-chargedanion population are reacted by adjusting the DC voltage offsets of thesegmented linear ion trap so as to store both the multiply-chargedpositive ions with the singly charged anions to facilitate the PTRprocess. The reagent anions are chosen such that, in this instance, thereagent anions behave as a Brønsted-Lowry base and such that theprecursor ions behave as one or more Brønsted-Lowry acids. The reagentanions are formed by separate ionization of a suitable reagentgas/liquid with sufficient vapor pressure, that includes but is notlimited to sulfur hexafluoride, perfluoro-1,3-dimethyl cyclohexane,perfluorodecalin, and perfluoroperhydrophenanthrene. After allowing thereaction to proceed for a specified time, a supplementary AC voltage isapplied across electrodes of the ion trap so as to eject the reagentanions, thereby leaving product ions and, possibly, some residualprecursor ions within the ion trap.

In the opposite polarity experiment, multiply-charged anions derivedfrom proteins or other biomolecules can also be reacted withsingly-charged cations. A variety of sources can be employed to generatesingly-charged cations including electron, chemical, and electrosprayionization processes. These reactions follow the same reaction kineticsdescribed previously. Typical reagent cations have included pyridine,benzo(f)quinolone, and the noble gases argon and xenon. In addition,multiply-charged proteins of opposite polarity have also been reacted aswell as the multiply-charged anions from nucleic acids with themultiply-charged cations of proteins.

In step 314 of the method 300 (FIG. 3A), a mass spectrum is obtained ofthe product ions from the PTR process retained in the ion trap over afull range of m/z ratios of interest. The mass spectrum may be obtained,in known fashion, by detecting ions that are sequentially ejected fromthe 3D or linear ion trap in order of their m/z ratios. Alternatively,the ions may be directed to a different mass analyzer of the massspectrometer, such as a Time-of-Flight (TOF) mass analyzer or anOrbitrap™-type of electrostatic trap mass analyzer, to be analyzed withgreater accuracy or mass resolution then may be available by sequentialscanning of the ion trap. Further, by directing the product ions to aseparate analyzer, the ion trap may be re-filled with a new sample ofprecursor ions while the mass analysis is being performed. If theaccurate mass analyzer is of a type—such as an FT-ICR mass analyzer oran Orbitrap™ mass analyzer—that detects image currents produced bycyclic ion motion within an ion trap, then the PTR reaction steps mayadvantageously reduce collision cross sections of targeted protein orpolypeptide molecules such that these molecules remain stable in thetrap for a sufficient length of time to generate high-quality massspectra. Also, the PTR product ions will have less kinetic energy whenleaving the high pressure C-trap region upon their transfer to theOrbitrap™ mass analyzer. Due to the PTR process, the resulting production population will be fully desolvated which will improve the qualityof the resulting mass spectrum.

In step 316 of the method 300, the mass spectrum generated by the massanalysis performed in step 314 is automatically examined so as torecognize one or more individual series of related m/z ratios, whereineach m/z ratio of a series represents a respective different chargestate—that is, a different degree of protonation—of a single intactprotein or polypeptide molecule. For example, see FIG. 9C which depictstwo different series of lines, represented by the envelope 905 and theenvelope 906, respectively. After ionization as well as subsequent tothe PTR reaction, each protein or polypeptide molecule, M, of massm_(p), is represented as at least one (and likely several different)protein or polypeptide cation species. Each such cation species of arelated series formed from the particular molecule, M, may berepresented by the chemical formula (M+zH)^(z+), where the integer, z,is the number of protons adducted to the original molecule or is thenumber of protons remaining on the protein after the PTR step. In thisexample, considering only monoisotopic ions, the mass-to-charge ratio,(m/z)_(ion), is thus given by:

(m/z)_(ion)≈(m _(p) +z×1.007)/z≈(m _(p) +/z≈m _(p) /z  (Eq. 1)

where the final approximation results from the fact that m_(p)>>z.Accordingly, such series of ion species representing only differentstates of protonation may be readily recognized by using automatedsoftware in real time to determine the monoisotopic ions. Once suchseries have been recognized, the molecular mass, m_(p), of the parentprotein or polypeptide molecule may be discerned in real time. Similarapproaches can be applied to larger molecular weight molecules usingaverage or monoisotopic mass as well.

The m/z values generated by the PTR process or, alternatively, themolecular weights obtained from the PTR product ions can then besearched against a database containing individual pathogen standardsthat contain the observed m/z values or molecular weights from knownreference standards/patient samples. By matching these m/z values ormolecular weights from a database containing individual referencedpathogens, a small subset of possible pathogen identifications isobtained. The subset can be limited by determining a particular massaccuracy, weighting the intensities of the individual peak, and/or byweighting the molecular weight values by mass in a given scoring system.This is illustrated in step 402 of FIG. 3A. In certain cases, the m/z ormolecular weight matches may provide a direct match to a particularpathogen identification. However, in all probability, the m/z molecularweight information will reduce substantially the number of possiblepathogen identifications that can be unequivocally identified usingtandem mass spectrometry. This process was originally described for usein conjunction with the steps 302, 304, 306, and 308-310 ininternational (PCT) patent application publication WO 2013/166169 A1.Additionally, Bayesian, logistic regression, or decision tree basedmethods can be employed to further refine the identification of thepathogen. In a preferred embodiment, this m/z or molecular weight searchis performed in real time during data acquisition (i.e., as the sampleis being analyzed). Alternatively, the search may be performedpost-acquisition (i.e., after the sample has been analyzed) as well. Thecomparison of a small number of m/z values or molecular weights (3-10)of proteins to a reference database will generally be sufficient tosignificantly reduce the candidate number of pathogen identifications tofive or less. This is illustrated is step 404 of FIG. 3A.

FIG. 3B schematically illustrates a flow diagram of a second exemplarymethod, method 370, in accordance with the present teachings. The steps302-314 of the method 370 (FIG. 3B) are identical to the similarlynumbered steps of the method 300 (FIG. 3A) and thus the description ofthese steps is not repeated here. The method 370 differs from the method300 only with regard to the steps following the generation of a massspectrum in step 314. According to the earlier-described method 300,this mass spectrum of PTR product ions is assumed to be sufficient todetect or quantify proteins and polypeptides of interest. However, inmany cases, it may be necessary to perform tandem mass spectrometry(sometimes referred to as MS/MS or MS^(n)) after the generation of PTRreaction products in order to resolve remaining ambiguities in therecognition of specific protein or polypeptide molecules. In suchsituations, the PTR reaction products may be considered to comprise afirst generation of reaction products which are then fragmented to forma second generation of product ions. The combination of a specific m/zratio of a first-generation reaction product with one or more specificm/z ratios of fragment ions may, in many cases, allow identification ofa specific protein or polypeptide molecule associated with a givenpathogen. In many instances the protein identified with a specificpathogen may also be found in other similar pathogens. In order tocorrectly identify a single pathogen, method 370 (specifically tandemmass spectrometry) may need to be performed on as many proteins that arepresent in a given PTR fraction, or multiple PTR fractions of the samesample.

Accordingly, steps 318-322 of method 370 (FIG. 3B) represent theapplication of the techniques of tandem mass spectrometry or selectedreaction monitoring (SRM) as applied to the ions formed by PTR. If theparticular employed mass spectrometry system permits, a portion of thePTR product ions may have already been stored (immediately after step312) in an ion storage apparatus of the mass spectrometer system. Insuch cases, the branching step 315 causes execution of step 317 a, inwhich the previously stored ions are retrieved for further processing.Otherwise, if the prior batch of PTR product ions was exhausted by themass analysis step (step 314), then, in accordance with the alternativestep 317 b, the steps 308-312 may need to be re-executed in order togenerate a new batch of such PTR product ions.

In step 318 of the method 370, certain of the PTR reaction-product ions(i.e., the first-generation product ions) within a particular m/z rangeor particular m/z ranges are mass isolated by ejecting ions whose m/zratios are not within the range or ranges of interest. The isolated ionsare subsequently fragmented in step 320. The particular chosen range orranges will generally be responsive to the details of a particularidentified charge-state sequence identified an immediately priorexecution of step 316 and the choice will generally be madeautomatically by computer. Thus, the choice of a particular m/z range orranges for isolation and fragmentation is an example of so-called“data-dependent analysis” (or “data-dependent acquisition”, etc.).

In most conventional MS/MS analyses, involving low-mass molecules of afew hundred to a few thousand Daltons, data-dependent fragmentationcomprises choosing the “top P number of the most abundant precursors”for tandem mass analysis based on the information of a preceding MS¹data acquisition, where the number P is either a constant or perhaps avariable input by a user. It has been found that this conventional formof data-dependent analysis does not perform well when used in theanalysis of multicomponent samples of biopolymer analytes. For example,FIG. 7C illustrates two charge state distributions, denoted by theenvelope 905 and the envelope 906, respectively. In this example, eachenvelope corresponds to a different respective analyte molecule species.Thus, the sets of lines encompassed by envelopes 905 and 906 may bereferred to as “molecular-species-correlative charge-statedistributions”. Considering the lines (individual m/z values) in FIG. 7Cto represent precursor ions, then if P=10, the conventionaldata-dependent fragmentation technique would choose the ten leftmostsolid vertical lines under the envelope 906 for fragmentation. Using theconventional technique, none of the dotted lines corresponding toenvelope 905 would be chosen. The conventional procedure would thusyield redundant information relating to the molecule speciescorresponding to envelope 906 and no information relating to themolecule species corresponding to envelope 905.

To overcome the shortcomings of conventional data-dependentfragmentation when applied to high-molecular-weight molecules, theinventors have developed the herein-used novel “top P uniqueanalyte-specific clusters” data-dependent technique so as to replace,for application to high-molecular-weight molecules, the previous “top Pnumber of the most abundant precursors” logic. Eachmolecular-species-correlative charge-state distribution is a set ofrelated mass spectral lines (m/z values) which are interpreted,according to the novel “top P unique analyte-specific clusters” logic,to all be generated from a single unique molecule. Eachmolecular-species-correlative charge-state distribution groups togethervarious charge states and isotopic clusters that are indicated to havebeen generated from a single molecule, prior to ionization. However, themolecular-species-correlative distribution excludes adducts, which areremoved prior to data analysis. According to the novel method,fragmentation is performed only on one (or possibly more) selectedrepresentatives of a given molecular-species-correlative charge statedistribution envelope thereby avoiding the redundancy noted aboveassociated with the conventional data-dependent fragmentation method.According to the novel “top P unique analyte-specific clusters” logic,after a representative m/z ratio (or ratios) has been chosen for a firstmolecular-species-correlative charge-state distribution, any furtherfragmentation is directed to a representative m/z ratio of the nextdetermined molecular-species-correlative charge-state distribution, andso on.

As previously described, the isolation performed in step 318 of themethod 370 may be accomplished by applying a supplemental AC voltageacross pairs of electrodes of an ion trap such that ions having m/zratios that are not within the range or ranges of interest are ejectedfrom the trap while those ions having m/z ratios that are within therange or ranges are retained within the trap. In some instances, the iontrap used for mass isolation may be identical to the mass analyzer usedto conduct the full-scan mass analysis in step 314.

The supplemental AC voltage applied to the ion trap used for massisolation may comprise a summation of superimposed frequencies such thations within two or more non-contiguous m/z ranges are simultaneouslyisolated. In the subsequent step 320, the mass-isolated first-generationproduct ions are fragmented by a suitable ion fragmentation technique,such as collision induced dissociation (CID). The fragmentation may beaccomplished by transferring the first-generation product ions (productions formed by PTR of original precursor ions), in known fashion, to adedicated fragmentation cell within which the transferred ions arefragmented so as to generate fragment ions, these fragment ionscomprising a second generation of reaction products. Optionally, aportion of the fragment product ions may be stored for possible futureadditional fragmentation in optional step 321.

In step 322 of the method 370 (FIG. 3B), the fragments generated in step320 are mass analyzed by a mass analyzer of the mass spectrometer. Ifthe second-generation product ions are produced within a fragmentationcell that is specifically dedicated for the purpose of fragmentation,the ions must be first transferred to the mass analyzer prior to theexecution of step 322. An ion trap mass analyzer may be employed toanalyze the second-generation product ions in step 322, in which casethe mass analyzer employed for step 322 may be identical to the massanalyzer employed to conduct the full-scan mass analysis of step 314.Alternatively, an accurate-mass analyzer capable of measuringmass-to-charge ratios to an accuracy of 10 ppm or better—such as anFT-ICR mass analyzer, a time-of-flight (TOF) mass analyzer or anOrbitrap™-type of electrostatic trap mass analyzer—may be employed forstep 322.

As is known, the correlation between the m/z value of a certain selectedion species subjected to fragmentation and the m/z value (or values) ofone or more fragment ion species produced by the fragmentation may besufficient to automatically determine (in step 402 b) the chemicalidentity of the selected ion species. In this case, the selected ionspecies is a PTR reaction-product species generated in step 312 that ismass-isolated in step 318. The identification of a small number (i.e.,3-10) of such proteins will generally be sufficient to uniquely identifya microorganism species (optional step 404 b). However, a single stageof fragmentation may be insufficient for performing a chemical speciesidentification. In such instances, the second generation product ionsmay be further fragmented so as to form a next generation of productions, indicated by the optional repeat (indicated with dashed lines)from step 322 back to step 318 in which a selected subset of thefragment product ions are isolated, according to their m/z values, andthe so-isolated fragment ions are further fragmented. More generally, asubset of the n^(th) generation of product ions may be selected forfurther fragmentation by any suitable ion fragmentation method such as,but not limited to, collision-induced fragmentation, higher-energycollisional dissociation, electron transfer dissociation, electroncapture dissociation, negative electron transfer dissociation,electron-detachment dissociation, in-source fragmentation,surface-induced dissociation, or photodissociation, whereby an(n+1)^(th) generation of product ions is formed. The results of the massanalysis step 322 may form the basis of an automated decision as towhether or not each additional fragmentation is required and, if so,which m/z values correspond to the ion species to be fragmented.

The method 300 diagramed in FIG. 3A, which was discussed above, providesa relatively simple and straightforward method of sample analysis thatmay be applicable for samples of relatively low complexity as, forexample, when highly-resolved chromatographic separation (step 306) hasbeen performed prior to introduction of a chromatographic fraction intoa mass spectrometer (step 308). However, the simple method 300 may notbe appropriate for more complex samples and the analysis of such samplesmay present a number of challenges. Firstly, the proteins present in acomplex mixture have a wide range of molecular weights. Secondly, thelarge number of charge states that result from the presence of a largenumber of lysine, arginine, histidine residues may result in multipleoverlapping sets of peaks, each set of peak corresponding to a differentchemical species. Thirdly, if the mass analysis (step 314) is ofsufficiently high resolution, the presence of resolved peaks of anisotopic distribution for any given charge state can confound most dataprocessing algorithms. Finally, the distribution of available ions amongmultiple charge states and, possibly, among multiple isotopic statesnecessarily reduces the signal intensity of any resolved peak in themass analysis.

In order to address the above-noted challenges in the analysis ofcomplex samples, the method 380, for which a schematic flow diagram isillustrated in FIG. 3C, provides the opportunity for conducting multiplePTR stages. Under the earlier-described method 300, it is assumed thatthe mass spectrum obtained (in step 314) of the first-generation PTRreaction products (generated in step 312) exhibits sufficientimprovement in signal-to-noise ratio and sufficient reduction inisobaric interferences such that charge-state sequences may berecognized and that proteins or polypeptides may be identified. If suchimprovement in mass spectral quality remains inadequate for suchpurposes after a first PTR reaction event, then the additionalrefinement steps 327-330 of the method 380 (FIG. 3C) may be performed.Further, one or more of the PTR stages may utilize the known techniqueof “ion parking” in order to simplify the charge state distribution, asnoted in the previous paragraph. Ion parking is a technique wherebyspecific selected ion/ion reactions within an ion trap are inhibited. Inpractice, a resonance excitation waveform is applied across electrodepairs of an ion trap, ion guide or other ion storage device in anamplitude that is insufficient to cause ion ejection but sufficient toincrease the velocities of ions having selected m/z values. Thisexcitation process increases the relative velocity between the excitedions (cations, for purposes of the present discussion) and reagentanions and it is believed that this relative velocity increase causes areduction in the rates of reaction between the excited cations andreagent anions.

During the PTR process, the rate of reaction between cations and reagentanions varies as the square of the charge number of the various cationswith the anion charge on the reagent ions equal to −1. Thus, in theabsence of ion parking, the PTR process leads to a rapid reduction inthe number of highly charged cations. Over the course of the reaction,the distribution of charge states of cations derived from a singlemolecular species, M (a protein or polypeptide molecule having massm_(p)), shifts towards lower charge states. The population of each ionspecies having an intermediate charge state will first increase as themore-highly-charged precursor ions lose protons and then decrease aseach respective species loses more protons then it gains from thediminishing quantity of more-highly-charged cations. The ultimateresult, if the PTR reaction is allowed to proceed to completion, iscomplete neutralization of all such cations and total loss of all massspectrometric signal.

When the ion parking technique is applied during the PTR reaction, thenthe charge reduction process is essentially stopped at the charge state,z₁, corresponding to the particular mass-to-charge ratio (for example,m_(p)/z₁) of the ions which are resonantly excited by the applied ACwaveform. Those precursor cations derived from the molecular species, M,with initial charge states, z, such that z>z₁ will lose protons untiltheir charge states are reduced to z₁, after which further reaction andproton loss will be inhibitied. Those precursor cations derived from themolecular species, M, with initial charge states, z, such that z<z₁ willbe completely neutralized. Accordingly, after PTR reaction with ionparking, a significant portion of the original protonated molecular ions(i.e., precursor ions) of molecule M will be represented, in a massspectrum, by the single ionic species having charge state, z₁. This“concentration” of the molecule species, M, into a single charge statecan advantageously amplify the mass-spectrometric signal associated withthat species, thereby improving signal-to-noise ratio and reducing thelower limit of detection and, optionally, the lower limit ofquantification of the species. Further, many isotopic variants of ionsgenerated from molecule species, M, will have m/z values outside of therange of values corresponding to the applied AC resonant excitationwaveform. Such isotopic variants will be neutralized so as to notinterfere with the mass spectrometric identification of ions ofinterest. Other isotopic variants comprise m/z values that are withinthe range of values corresponding to the applied AC resonant excitationwaveform. The isotopic distribution pattern of such isotopically variantions will be greatly simplified relative to the isotopic distributionobserved in the original precursor ions because they will mostly relateto the single charge state, z₁ of ions generated from molecule, M.

Returning to the discussion of the method 380 outlined in FIG. 3C, it isto be noted that the steps 302-310 of the method 380 are identical tothe similarly numbered steps of the method 300 (FIG. 3A) and are notre-described here. Subsequently, in step 328, precursor ions aresubjected to PTR, optionally as modified by the ion parking technique.As previously noted, step 328 is executed by applying a supplemental ACexcitation waveform across a pair of electrodes of an ion trap withinwhich sample-derived cations are reacted with reagent anions for apredetermined time period. As described above, the employment of this“ion parking” procedure will concentrate the distribution of ionsderived from any particular protein or polypeptide into a particularrestricted range of m/z values. This will generally restrict the ionsderived from any particular protein or polypeptide into a particularcharge state, thereby simplifying a resulting mass spectrum andincreasing the intensity of any mass spectral peaks corresponding to theparticular protein or polypeptide. The particular range of m/z valuesinto which the ions are restricted may comprise ions of differentrespective charge states derived from different respective molecularspecies. In some embodiments, the applied AC waveform used to effect theion parking may comprise a summation of waveforms of differentrespective frequencies such that the summed waveform causes the PTRreaction to yield a final population of PTR product ions correspondingto two or more non-contiguous m/z ranges.

In the subsequent step 330, the population of PTR product ions producedin step 328 is mass analyzed. Prior to this mass analysis, a portion ofthe PTR product ions may be stored (step 329) in preparation forpossible subsequent PTR reaction. Depending upon the results of the massanalysis of the PTR product ions, an automatic decision may be made tosubject the PTR product ions to such further PTR reaction, as indicatedby the dashed line optional pathways shown in FIG. 3C. The decision mayalso be made, based on the results of the mass analysis, to only subjecta selected subset of the PTR product ions to subsequent PTR reaction. Insuch cases, step 327 is executed. If the mass analyzer employed in step330 is of a type that detects image currents produced by cyclic ionmotion within an ion trap or other ion storage device—such as an FT-ICRmass analyzer or an Orbitrap™ mass analyzer—then the PTR reaction stepsmay advantageously reduce collision profiles of targeted protein orpolypeptide molecules such that these molecules remain stable in thetrap for a sufficient length of time to generate high-quality massspectra. After a sufficient number of PTR reaction steps, the chemicalidentity of the protein or polypeptide may then be rapidly discerned (instep 402 c) by matching to databases of known molecular masses. Theidentification of a small number of (3-10) of proteins will generally besufficient to uniquely identify a microorganism species (optional step404 c). Identification can also be accomplished via the use ofclassifiers applied to the PTR data as discussed previously thatincludes but is not limited to Bayesian, logistic regression or decisiontree based approaches.

FIGS. 3D-3E illustrate, in flow diagram form, another method, method390, in accordance with the present teachings. The steps 302-330 of themethod 390 are shown in FIG. 3D and are identical to thepreviously-discussed similarly-numbered steps of the method 380 (FIG.3C); thus, these steps are not re-described here. Instead of proceedingto the identification step 402 d directly from step 330 (as in themethod 380 of FIG. 3C), execution of the method 390 (FIGS. 3D-3E)proceeds from step 330 to a mass selection and isolation step 332. Instep 332, a subset of the PTR product ions—generated by one or moreapplications of the PTR procedure—are isolated according to selected m/zratios. Decisions regarding the specific m/z ratios to be isolatedduring this step may be automatically performed based on the massspectrometric results obtained in step 330. The steps 332-338illustrated in FIG. 3E represent an ion fragmentation procedure whichmay be iterated so as to produce multiple generations of fragmentationproduct ions. These steps 332-338 are similar to the steps 318-322 ofthe method 370 illustrated in FIG. 3B and are thus not discussed indetail.

After execution of the fragmentation and mass analysis steps, thepeptide identification step 402 d of the method 390 (FIG. 3E) isexecuted. Whereas the identification step 402 a of the method 300 (FIG.3A) makes use only of the m/z ratios (or molecular weights) of ionspecies comprising protonated or multiply-protonated analyte molecules,the identification step 402 d of the method 390 also takes into accountthe m/z ratios of the fragments—possibly of multiple generations—ofthese ion species. Thus, in the case of complex mixtures of proteins orpolypeptides, a greater confidence may be associated with the results ofthe identifications made using the method 390. Control of theexperiments may be performed in real time according to some embodimentsby making use of real-time data deconvolution as noted above. Theidentification of a small number of (3-10) of proteins species in step402 d will generally be sufficient to uniquely identify a microorganismspecies in step 404 d.

FIG. 3F diagrammatically illustrates, in flow diagram form, anothermethod, method 395, in accordance with the present teachings. Most ofthe steps in the method 395 (FIG. 3F) are similar to similarly numberedsteps in the method 370 (FIG. 3B) and these steps are not re-describedin detail. Similarly to the method 370, the method 395 includes a step(step 312) of subjecting original precursor ions to PTR charge reductionfollowed by steps (steps 318 and 320) of isolating selected PTR production species and subjecting the isolated ion species to fragmentation soas to form fragment product ion species. The method 395 differs frommethod 370 through the provision of an additional step, step 340, ofsubjecting the fragment ions to PTR charge reduction. Since the variousPTR product ion species generated from the original precursor ions maybe multiply-charged and may be distributed among species with variousdegrees of protonation, the fragment ions formed from them maythemselves be distributed among multiple protonation states. The PTRcharge reduction of the fragment ion species in step 340 can simplifythe charge state distributions of the fragment ions prior to their massanalysis in step 341. Optionally, any of the PTR steps (step 312 andstep 340) may employ ion parking.

Example A

FIGS. 4A and 4B provide an example of mass spectroscopic signalenhancement provided by a single PTR reaction step (e.g., as in themethod 300 shown in FIG. 3A). In a first application (FIGS. 4A, 4B), anextract from the pathogen E. coli was analyzed via direct infusion; themass spectrum of the first-generation electrospray-generated ions isshown in FIG. 4A. As expected, there are many proteins present thatoverlap at various m/z values leading to the presence of a broadspectral region between approximately m/z=780 and m/z=1420 within whichmany ions are detected but with very little usable information in termsof discernible protein charge state distributions. Next, an m/z “window”of the first-generation ions of width 2 Th and centered at m/z=750 wasisolated and the resulting isolated ion population was subjected to PTRreaction. The m/z position 412 a shown in FIG. 4A indicates the centerposition of the isolation window.

FIG. 4B shows a mass spectrum of the PTR reaction products of precursorions of the E. coli extract. The PTR reactions were carried out withreagent anions derived from 3 ppm of sulfur hexafluoride (SF₆) in anitrogen gas stream delivered to a glow discharge reagent ion sourcecontained within the ion optics of a mass spectrometer of the samegeneral configuration as illustrated in FIG. 2. As with most PTRproduct-ion spectra, the mass spectrum shown in FIG. 4B exhibits arelatively intense isolated peak at the position (indicated as position412 b) of the original first-generation-ion isolation window. Such peaksat the position of the isolation window generally indicate the presenceof residual singly-charged first-generation ions—generally not ofinterest—that fortuitously occur at the position of the isolation. Otherpeaks in the spectrum of FIG. 4B represent product ions generated fromthe PTR reaction. These product ions generally comprise overlapping setsof related ions, each set corresponding to ions comprising adistribution of charge states from an original multiply-chargedprecursor ion within the original isolation window. One such potentialcharge-state distribution pattern is approximately indicated by theenvelope 413. The results shown in FIGS. 4A and 4B show that the PTRreaction process generally significantly simplifies the spectrum andreduces background interference. Nonetheless, since many protein-derivedor peptide-derived precursor ions may be present in the originalisolation window, the charge-state distribution patterns may overlap.Mathematical decomposition (sometimes referred to as “deconvolution”)may be required to recognize the individual patterns.

Example B

FIGS. 5A and 5B illustrate an example of analysis of an E. Coli extractthat is performed by a procedure that includes two stages of PTRreaction (for example, see steps 327, 328, 329 and 330 of method 380 inFIG. 3C). FIG. 5A illustrates a PTR product ion spectrum generatedisolated first-generation precursor ions from within a 5 Th mass windowcentered at m/z=1200, indicated by position 711 in FIG. 5A. In thisinstance, the initial PTR spectrum does not include peaks that aresufficiently well resolved to enable identification of any proteins inthe sample. Therefore, a subset of the first-generation PTR product ionswere isolated for a second stage of PTR from within a 5 Th mass windowcentered at m/z=1320, indicated by position 712 a in FIG. 5A andposition 712 b in FIG. 5B. The second-generation PTR product ions, whichoccur at m/z ratios greater than 1320 in FIG. 5B show clear charge-statedistribution patterns that may be successfully used for identificationof proteins in the sample.

FIGS. 6A-6G illustrate an example of analysis of an E. coli extract thatis performed by a procedure that includes a first stage of product ionformation by PTR reaction followed by subsequent stages of CID of thePTR reaction product ions (for example, see steps 312 through 322 method370 in FIG. 3B). FIG. 6A illustrates a PTR product ion spectrumgenerated isolated first-generation precursor ions from within a 5 Thmass window centered at m/z=640, indicated by position 811 in FIG. 6A.The PTR product ions occur at m/z ratios greater than that indicated byposition 811 in FIG. 6A. The three most intense PTR product ions,located at m/z ratios of 833, 926 and 917 and indicated by mass spectralpeaks 813, 814 and 815, respectively, in FIG. 6A, were then individuallyisolated and separately subjected to collision-induced dissociation soas to produce three sets of second-generation product ions. FIGS. 6B and6C respectively depict the isolated PTR product ion at m/z=833 and thesecond generation product ions (fragment ions) generated by CID of theisolated PTR product ion. Likewise, FIGS. 6D and 6E respectively depictthe isolated PTR product ion at m/z=926 and the second generationproduct ions generated by CID of the isolated PTR product ion atm/z=926. Likewise, FIGS. 6F and 6G respectively depict the isolated PTRproduct ion at m/z=917 and the second generation product ions generatedby CID of the isolated PTR product ion at m/z=917.

Example C

As should be evident from the previous discussions, positive ionelectrospray ionization of any protein or polypeptide molecule willproduce a plurality of ions comprising different respective chargestates (i.e., number of charges) as a result of different degrees ofprotonation of the original molecule. Charge states of +50 or more orpossible and each charge state will be represented by multiple massspectral lines representing different degrees of natural isotopicsubstitution. A further complication arises from the fact that for mostnatural biological samples, numerous different proteins of polypeptidemolecules may be represented in a mass spectrum. A yet furthercomplication arises from the fact that many other molecules—notnecessarily of interest—may be present in a sample.

In many basic-research-oriented studies, the above-noted complicatingfactors of multiple analytes and multiple interfering species may bepartially or wholly resolved by performing chromatic separation prior tointroducing each separated compound individually into a massspectrometer. However, clinical analyses may often be performed undertight time constraints that do not allow for traditional time-consumingchromatographic separation. The clinical time constraints may only allowfor an incomplete or partial separation using either solid-phaseextraction (SPE), size-exclusion chromatography, or the method of FastPartial Chromatographic Separation (FPCS) described above. Thus, whensuch partial separation procedures are employed, the mass spectralsignature of any particular protein or polypeptide may be spread outover a wide mass-to-charge ratio and may be complexly overlapped withthe mass spectral signatures of other compounds. Since the availablecharge, as provided by an electrospray apparatus, will be spread outover many different types of ions, most of the observed mass spectrallines will coexist with and possibly be hidden within a general denselypopulated and low-intensity or ill-defined spectral “background”indicated schematically by spectral envelope 902 in FIGS. 7A-7B.

The inventors have realized that the mass spectral signature of anyparticular protein, polypeptide or other biologically relevanthigh-molecular-weight analyte may be hypothetically amplified bysimultaneously isolating multiple charge states of the same originalmolecule and then reacting the assemblage of multiple charge states withPTR reagent ions so as to simultaneously reduce the assemblage to asmall number of charge states distributed over a few charge-statevalues, these charge-state values being reduced relative to the originalcharge states. This concept is illustrated by the vertical boxes 904a-904 g shown overlaid over the general charge-state envelope 902 inFIG. 7A. Each such vertical box represents a particular precursor ionspecies and represents a small range of m/z values chosen to correspondto a particular charge state (and possibly including a few isotopicvariants) of a particular analyte. Hypothetically, if all ions outsideof the ranges corresponding to the vertical boxes could be excluded andonly the ions from within the indicated ranges mixed together, thensubsequent PTR would essentially provide a summation of the signals fromthe various original plurality of charge states. The use of suchmulti-species isolation of a plurality of precursor ion species canincrease the sensitivity of the analysis up to N-fold, where N is thenumber of m/z ranges selected and simultaneously isolated.

Such multiple-species isolation is fairly easy to achieve when isolationis performed in a linear ion trap (such as the low-pressure linear trapcell 217 b illustrated in FIG. 2), because resonance-excitationwaveforms, which are used to eject unwanted ions, may be constructedwith multiple notches. Each such notch corresponds to a differentrespective m/z window within which ions will not be ejected (and thusisolated). Thus, the co-isolating of a plurality ofelectrospray-generated (first-generation) precursor ion species may beperformed, in some embodiments, by simultaneously isolating all of theplurality of precursor ion species. One way of doing this is by applyinga broadband resonance ejection frequency waveform to an ion trap intowhich ions received from an electrospray source have been introduced,wherein the waveform comprises multiple summed sinusoidal frequencycomponents, wherein included frequency components corresponding to them/z ranges of ions that one desires to eject from the trap and excludedfrequency components correspond to the m/z range of ions that onedesires to retain within the trap. In this procedure, the omittedfrequencies define one or more frequency notches in the ejectionfrequency waveform. The frequency components may be calculated by firstchoosing a desired multi-notch waveform and then calculating an inverseFourier Transform of the desired waveform.

Alternatively, the co-isolating of the plurality of precursor ionspecies may be performed by isolating individual precursor ion speciesin a conventional sense, one ion species at a time using a respectivesingle-notch waveform applied to an ion trap. The individually isolatedprecursor ion species may be transferred, one at a time, to an ionstorage component (such as the multipole ion guide 214 illustrated inFIG. 2) in which the various selected and isolated ion species areaccumulated over time. As a yet-further alternative, the co-isolating ofthe plurality of precursor ion species may be performed by passing aplurality of ions received from an electrospray source through aquadrupole mass filter while the bandpass of the quadrupole mass filteris sequentially tuned to preferentially transmit, in turn, each m/zrange corresponding to a particular precursor ion species. The filteredions that pass through the quadrupole mass filter are then passed intoan ion storage component that accumulates the ions from all thepreferentially transmitted m/z ranges. For example, in the massspectrometer 150 a illustrated in FIG. 2, the quadrupole mass filter 208may perform the sequence of filtering steps and the ions of eachtransmitted m/z range may be transmitted into and accumulated within themultipole ion guide 214. The accumulated precursor ion species may thenbe transferred back to the low-pressure cell 217 b for PTR reaction.

The above-described procedure employing simultaneous multi-speciesisolation assumes that appropriate isolation ranges 904 a-904 g a prioriknown. Such knowledge about the correct isolation ranges to employ maybe available in certain instances of targeted analysis, when theidentity of (and other information pertaining to) an analyte that is tobe searched for is already known and the purpose of the analysis is todetermine the presence or absence of the analyte or to determine thequantity or concentration of the analyte. However, the above assumptionmay be invalid in the case of survey analyses, in which the identitiesof analytes may not be known in advance. In such latter cases, aninitial random survey may be performed by isolating a random mass range903 of the first-generation ions, as schematically depicted in FIG. 7B,and then reacting the isolated ions with a PTR reagent anion. Aspreviously illustrated in FIGS. 4A and 4B, such a procedure can provideresolved, interpretable mass spectral lines relating to charge statedistributions of one or more analytes. In many instances, a set ofrelated lines may be recognized with by the mutual consistency of theirm/z values with Eq. 1 for a certain sequence of consecutive integers, z.The degree of consistency of the line positions may be performedautomatically, by computer analysis, such that overlapping sets of suchrelated lines may be mathematically decomposed and recognized.

As an example of the above type of analysis, mathematical decompositionof the PTR product ion lines generated by isolation and reaction ofprecursor ions within m/z range 903 may lead to recognition of twooverlapping sets of lines, depicted by envelope 905 and envelope 906, asillustrated in FIG. 7C. With the information provided by this initialsurvey procedure, an appropriate and consistent set of m/z values may bechosen, which may be employed in a subsequent simultaneous multi-speciesisolation and reaction procedure. For example, the m/z values of certainresolved instances of the lines under envelope 905 may be chosen,perhaps automatically. Subsequent multi-species isolation and PTRreaction of precursor ions corresponding to these chosen m/z values willthen provide an amplified spectrum that may be employed to determine aquantity or concentration of the particular molecule represented by theenvelope 905. This procedure may later be repeated using the associatedwith envelope 906 so as to determine a quantity or concentration ofanother molecule. The determined quantities or concentrations may not beaccurate, in an absolute sense, but the ratios of the determinedquantities or concentrations may provide useful information relating torelative quantities or concentrations. This entire procedure outlinedabove may be repeated multiple times using different randomly chosen m/zranges 903, thereby providing determinations of relative quantities orconcentrations of several compounds. As stated previously, control ofsuch experiments can be accomplished in a data-dependent fashionutilizing the results of real-time spectral deconvolution.

FIG. 8 provides a general flow diagram of an exemplary method, method397, of survey analysis using the above-outlined PTR signalamplification by reaction of PTR reagent ions with first-generation ionsfrom multiple non-contiguous m/z ranges. Steps 302, 304, 306, 308, 309,312, 314, 316, 402 and 404 of the method 397 are identical to thesimilarly numbered steps of the method 300 illustrated in FIG. 3A andare thus not re-described here. Also, the new step 311 is similar to thepreviously described step 310 of method 300, except that step 311 refersonly to mass isolation of a random m/z range (such as the range 903depicted in FIG. 7C) of first-generation ions, instead of to a “randomor predetermined m/z range or ranges” as described for the prior method300. After the initial survey PTR reaction (step 312) and identificationof charge-state sequences (step 316), the step 323 is executed, in whichmultiple non-contiguous m/z ranges of the first-generation ions areisolated and accumulated, wherein the non-contiguous m/z rangescorrespond to an identified charge state sequence. The first-generationions may be obtained from a previously stored batch of such ions (priorstep 319 a) or, alternatively (prior step 319 b), the sampleintroduction and electrospray ion generation step may need to berepeated.

After the isolation and accumulation of multiple non-contiguous m/zranges of the first-generation ions (step 323), the accumulated ions arereacted with PTR reagent ions (step 324). The resulting amplifiedspectra will generally be of high quality thereby facilitating thederivation (step 325) of, for example, an accurate molecular weight ofthe molecule corresponding the multiple non-contiguous m/z ranges or anaccurate quantity, concentration, or relative abundance of suchmolecule. If an immediately prior execution of step 316 identified morethan one set of related m/z ratios, then step 319 a or 319 b and steps323-325 may be executed again (following the leftmost “Y” branch of step326) using a new set of non-contiguous m/z ranges that correspond to adifferent identified charge state sequence. If a search for possibleadditional analytes is to be continued, then execution may return tostep 311 (following the rightmost “Y” branch of step 326) at which adifferent random m/z range is chosen.

Example D

According to another method for reduction of sample complexity utilizingproton transfer reactions in accordance with the present teachings, massspectrometric analysis employing PTR can be coupled directly withchromatography in order to simplify and detect additional proteins thatwould otherwise be missed. In this embodiment, a full scan mass spectrumis taken and the protein molecular weights are calculated using areal-time deconvolution program. Next, an isolation window is chosen ofa defined width and the subset of m/z values in the window are subjectedto PTR reactions.

For example, FIG. 9A shows a full scan mass spectrum of first-generationions generated from eluate at a retention time of 10 min. and 30 s.during the course of a ten-minute gradient reverse-phase liquidchromatography separation of an E. coli extract. As indicated by thebraces in FIG. 9A, this full-scan mass spectrum exhibits the distinctspectral signatures of two proteins having approximate molecular weightsof 35.1 and 31.1 kDa respectively. For the next step, a population ofions having m/z values within an m/z isolation window 510 of 10 Th widthand centered at 750 Th were isolated. The isolated ion population wasthen subjected to PTR reactions with the anionic reagent sulfurhexafluoride for 10 ms. The resulting product ion mass spectrum, shownin FIG. 9B, exhibits the mass spectral signatures of two additionalproteins not seen in the full-scan mass spectrum having molecularweights of 11220.07 Da and 24599.56 Da. In addition, the 35.1 kDaprotein component previously observed in the full-scan mass spectrumalso exhibits a spectral signature in the PTR product ion spectrum whichincludes a line, outlined in box 520, corresponding to a +47 chargestate at a nominal m/z value of 749. The line at 749 Th representscharge reduction of even-more-highly-charged states of the 35.1 kDaprotein. The proteins observed at 11.2 and 24.6 kDa would not otherwisebe identified in the absence of the PTR step in this example of areverse-phase chromatographic run as a result of complex spectraloverlap and interfering noise from an abundance of singly-chargedbackground ions.

FIGS. 10A and 10B show the results of a similar chromatography/MSexperiment obtained from eluate at a retention time of 42 min. and 30 s.from a sixty-minute gradient elution run. As shown in FIG. 10A, a highbackground at this elution time causes difficulty in identifying analytepeaks in the full-scan spectrum. However, the PTR product ion spectrumplotted in FIG. 10B is much more amenable to interpretation and massspectral deconvolution. The PTR product ion spectrum exhibits the massspectral signatures of three distinct proteins—specifically havingmolecular weights 11165.92 Da, 13480.28 Da and 18727.23 Da—that wouldnot otherwise be observed. In this instance, the PTR product ions weregenerated from isolated precursor ions generated from the mass spectralwindow, indicated by box 610 in FIG. 10A, of 10 Th width centered at m/z750. By performing this type of analysis upon eluates that elute atvarious different retention times during the course of a singleexperiment, a sufficient number of sample peptides may be recognized soas to enable identification of a microorganism to the species,subspecies, or strain level. As also indicated by the results shown inFIGS. 9A-9B, if there is m/z overlap of protein ions from the full massspectrum within the isolation window, then the protein will also be seenin the PTR product ion mass spectrum.

Interestingly, the full scan mass spectrum and PTR product-ion massspectrum can provide complementary information, as illustrated in FIGS.11A and 11B which represent mass spectral results obtained from eluateeluting at a retention time of 18 min. and 9 s. over the course of athirty-minute chromatographic separation. In this example, the full-scanmass spectrum (FIG. 11A) exhibits a strong mass spectral signature ofessentially a single protein having a molecular weight of 9534.3 Da,However, when a PTR product ion spectrum is generated from ions isolatedwithin a 10 Th wide window centered around m/z 750 Th (box 530), themass spectral signature comprises a strong signal from a protein havinga molecular weight of 14965.5 Da (best represented by the peak 535 ofthe +12 charge state at approximately 1247 Th) along with five otherminor proteins having molecular weights of 12669.8 Da, 14150.0 Da,14236.1 Da, 14965.5 Da, and 15117.5 Da. FIG. 11C is a full-scan massspectrum obtained from eluate eluting during the same chromatographicseparation at a retention time of 22 min. and 27 s. The spectrumincludes peaks indicating the presence of a protein having a molecularweight of 24961.3 Da. Upon PTR reaction of ions isolated within theisolation window 540, the PTR product ion spectrum shown in FIG. 11D wasobtained. The mass spectral signature in the PTR product ion spectrumincludes a relatively strong signal from a protein having a molecularweight of 28461.5 Da (best represented by the peak 545 of the +22 chargestate at approximately 1294 Th) as well as two other proteins havingmolecular weights of 18590.5 Da and 20168.0 Da. Thus, from just the dataat these two retention times, it is possible to detect the presence andthe molecular weights of eleven different proteins.

Additional Examples

The following paragraphs list additional specific examples of variousspecific embodiments in accordance with the present teachings.

Example 1

A method for identifying the presence or absence of a protein orpolypeptide analyte compound within a liquid sample comprising a mixtureof compounds that includes a plurality of protein compounds or aplurality of polypeptide compounds or pluralities of both protein andpolypeptide compounds, the method comprising:

-   -   (a) introducing a portion of the liquid sample into an        electrospray ionization source of a mass spectrometer;    -   (b) forming positively charged ions of the mixture of compounds        of the portion of the liquid sample by electrospray ionization,        the positively charged ions comprising a plurality of ion        species;    -   (c) isolating a first subset of the ion species comprising a        first mass-to-charge (m/z) ratio range that includes an m/z        ratio of a particular pre-determined multiply-protonated        molecular species of the analyte compound;    -   (d) generating a plurality of first-generation product ion        species from the isolated first subset of ion species by causing        the isolated first subset of ion species to be reacted, for a        predetermined time duration, with reagent anions that, upon        reaction, extract protons from each of one or more ion species        that comprises a protonated molecular species of a protein or        polypeptide compound;    -   (e) generating a mass spectrum, using a mass analyzer, of either        the first-generation product ion species or of second-generation        product ion species generated from the first-generation product        ion species;    -   (f) conducting a search of the mass spectrum of either the        first-generation or the second-generation product ion species        for a set of one or more m/z ratios that are diagnostic of the        protein or polypeptide analyte compound; and    -   (g) identifying the presence of the analyte compound within the        sample if the set of one or more m/z ratios is identified in the        mass spectrum.

Example 2

A method as recited in Example 1, further comprising repeating the steps(a) through (e) a second time, wherein the steps (f) and (g) areperformed during or prior to the second performing of the steps (a)through (e).

Example 3

A method as recited in Example 1, further comprising repeatedlyperforming steps (a) through (g) a plurality of times, wherein eachrepetition of step (a) comprises introducing, into the electrosprayionization source, an eluate from a chromatographic column correspondingto a respective retention time.

Example 4

A method as recited in Example 1, wherein the step (f) comprisesconducting a search of the mass spectrum of the first-generation production species for a series of m/z ratios that correspond to a sequence ofmultiply-protonated ion species of the analyte compound that areprogressively charge-reduced with respect to the charge state of theparticular predetermined multiply-protonated molecular species.

Example 5

A method as recited in Example 1, wherein:

-   -   the step (c) comprises further isolating a second subset of the        ion species comprising a second m/z ratio range that includes an        m/z ratio of a particular predetermined multiply-protonated        molecular species of a second protein or polypeptide analyte        compound;    -   the step (f) comprises conducting an additional search of the        mass spectrum of either the first-generation or the        second-generation product ion species for a second set of one or        more m/z ratios that are diagnostic of the second protein or        polypeptide analyte compound; and    -   the step (g) comprises identifying the presence of the second        analyte compound within the sample if the second set of m/z        ratios is identified in the mass spectrum.

Example 6

A method as recited in Example 5, wherein the first m/z ratio range isidentical to the second m/z ratio range.

Example 7

A method as recited in Example 5, wherein the step (c) comprisessimultaneously isolating the first subset of the ion species comprisingthe first m/z ratio and the second subset of the ion species comprisingthe second m/z ratio range such that the first and second m/z ratioranges are non-contiguous.

Example 8

A method as recited in Example 1, wherein the step (d) of generating aplurality of first-generation product ion species comprises causing theisolated first subset of ion species and reagent anions to be reactedfor a time duration that causes the product ion species to be stableagainst decomposition during the subsequent generation of the massspectrum in step (e).

Example 9

A method as recited in Example 8, wherein the step (e) comprisesgenerating a mass spectrum of the first-generation product ion speciesusing a mass analyzer that generates the mass spectrum by detectingimage currents caused by motions of the ions of the product ion specieswithin an ion trap.

Example 10

A method as recited in Example 1, wherein the step (d) of generating aplurality of first-generation product ion species includes applying asupplemental AC voltage across electrodes of an ion trap within whichthe isolated first subset of ion species are reacted with reagentanions, wherein a frequency of the supplemental AC voltage is such thation-ion reaction between the reagent anions and selectedfirst-generation product ion species is inhibited.

Example 11

A method as recited in Example 10, wherein the frequency of thesupplemental AC voltage is such that, subsequent to the execution ofstep (d), product ions formed from the analyte compound existsubstantially as a single ion species having a particular charge state.

Example 12

A method as recited in Example 11, wherein:

-   -   the step (e) comprises generating a mass spectrum of the        first-generation product ion species; and    -   wherein the mass of the single ion species is greater than        20,000 Da and the charge state of the single ion species is        sufficiently great such that ions of the single ion species may        be detected, during the generation of the mass spectrum, by        either a quadrupole mass analyzer, a Fourier transform ion        cyclotron resonance mass spectrometer or an electrostatic trap        mass analyzer.

Example 13

A method as recited in Example 1, wherein the step (e) of generating amass spectrum comprises generating a mass spectrum of second-generationproduct ion species, wherein the second-generation product ion speciesare generated by the steps of:

-   -   isolating a subset of the first-generation product ion species        comprising a particular product-ion m/z ratio range; and    -   fragmenting the isolated subset of the first-generation product        ion species so as to form fragment ion species, wherein the        fragment ion species comprise the second-generation product ion        species.

Example 14

A method as recited in Example 1, wherein the step (e) of generating amass spectrum comprises generating a mass spectrum of second-generationproduct ion species, wherein the second-generation product ion speciesare generated by:

-   -   causing the first-generation product ion species to be reacted,        for a second predetermined time duration, with the reagent        anions, wherein products of reaction between the        first-generation product ion species and the reagent anions        comprise the second-generation product ion species.

Example 15

A method as recited in Example 14, wherein a supplemental AC voltage isapplied across electrodes of an ion trap within which thefirst-generation product ion species are reacted with the reagentanions, wherein a frequency of the supplemental AC voltage is such thation-ion reaction between the reagent anions and selected product ionspecies is inhibited.

Example 16

A method as recited in any one of Examples 1-15, further comprisinggenerating the liquid sample comprising the mixture of compounds by aprocedure comprising:

-   -   (i) culturing microorganisms or cells;    -   (ii) lysing the cultured microorganisms or cells; and    -   (iii) extracting proteins from the lysate of cultured        microorganisms or cells.

Example 17

A method as recited in Example 16, wherein the step (iii) of extractingthe liquid sample from the lysate includes passing the lysate through asolid-phase-extraction apparatus.

Example 18

A method of identifying the presence or absence of a microorganism typein a sample, comprising:

-   -   (i) identifying a list of analyte compounds whose simultaneous        presence in the sample is diagnostic of the presence of the        microorganism type in the sample, said list of analyte compounds        comprising protein compounds, polypeptide compounds or both        protein and polypeptide compounds;    -   (ii) extracting, from the sample, a liquid solution comprising a        mixture of sample-derived proteins and polypeptides;    -   (iii) for each respective analyte compound in the list,        performing the steps of:        -   (a) introducing a portion of the liquid solution into an            electrospray ionization source of a mass spectrometer;        -   (b) forming positively charged ions of the mixture of            compounds of the portion of the liquid solution by            electrospray ionization, the positively charged ions            comprising a plurality of ion species;        -   (c) isolating a first subset of the ion species comprising a            first mass-to-charge (m/z) ratio range that includes an m/z            ratio of a particular predetermined multiply-protonated            molecular species of the respective analyte compound;        -   (d) generating a plurality of first-generation product ion            species from the isolated first subset of ion species by            causing the isolated first subset of ion species to be            reacted, for a predetermined time duration, with reagent            anions that, upon reaction, extract protons from each of one            or more ion species that comprises a protonated molecular            species of a protein or polypeptide compound;        -   (e) generating a mass spectrum, using a mass analyzer, of            either the first-generation product ion species or of            second-generation product ion species generated from the            first-generation product ion species;        -   (f) conducting a search of the mass spectrum of either the            first-generation or the second-generation product ion            species for a set of one or more m/z ratios that are            diagnostic of the respective analyte compound; and        -   (g) identifying the presence of the respective analyte            compound within the liquid solution if the set of one or            more m/z ratios is identified in the mass spectrum; and    -   (iv) identifying the presence of the microorganism type within        the sample if the presence of each and every analyte compound of        the list of analyte compounds is identified within the liquid        solution.

Example 19

A method of identifying the presence or absence of a microorganism typein a sample, comprising:

-   -   (i) identifying a list of analyte compounds whose simultaneous        presence in the sample is diagnostic of the presence of the        microorganism type in the sample, said list of analyte compounds        comprising protein compounds, polypeptide compounds or both        protein and polypeptide compounds;    -   (ii) extracting, from the sample, a liquid solution comprising a        mixture of sample-derived proteins and polypeptides;    -   (iii) introducing at least a first portion of the liquid        solution into an ionization source of a mass spectrometer;    -   (iv) generating, from the at least first portion of the liquid        solution at the ionization source, positively charged ions of        the mixture of compounds, the positively charged ions comprising        a plurality of ion species;    -   (v) isolating at least a first subset of the plurality of ion        species, each isolated subset of the at least a first isolated        subset comprising a respective mass-to-charge (m/z) ratio range;    -   (vi) generating a plurality of first-generation product ion        species from each isolated subset of ion species by causing each        said isolated subset of ion species to be reacted, for a        predetermined time duration, with reagent anions that, upon        reaction, extract protons from each of one or more ion species        of said isolated subset of ion species that comprises a        protonated molecular species of a protein or polypeptide        compound;    -   (vii) generating at least one mass spectrum, using a mass        analyzer of the mass spectrometer, of either first-generation        product ion species or second-generation product ion species        generated by further reaction of the first-generation product        ion species;    -   (viii) for each respective analyte compound in the list,        performing the steps of:        -   (a) conducting a search of the at least one mass spectrum of            either the first-generation or the second-generation product            ion species for a set of one or more m/z ratios that are            diagnostic of the respective analyte compound; and        -   (b) identifying the presence of the respective analyte            compound within the liquid solution if the set of one or            more m/z ratios is identified in the mass spectrum; and    -   (ix) identifying the presence of the microorganism type within        the sample if the presence of each and every analyte compound of        the list of analyte compounds is identified within the liquid        solution.

Example 20

A method as recited in Example 19, wherein a performing of the steps (a)and (b) is performed concurrently with the performing of one or more ofthe steps (iii) through (vii).

Example 21

A method as recited in Example 19, wherein the microorganism type isdefined as a particular genus of bacteria and the list of analytecompounds includes a sufficient number of analyte compounds that arediagnostic of the particular genus of bacteria to enable identificationof the presence or absence of the particular genus of bacteria in thesample.

Example 22

A method as recited in Example 19, wherein the microorganism type isdefined as a particular species of bacteria and the list of analytecompounds includes a sufficient number of analyte compounds that arediagnostic of the particular species of bacteria to enableidentification of the presence or absence of the particular species ofbacteria in the sample.

Example 23

A method as recited in Example 19, wherein the microorganism type isdefined as a particular sub-species of bacteria and the list of analytecompounds includes a sufficient number of analyte compounds that arediagnostic of the particular sub-species of bacteria to enableidentification of the presence or absence of the particular sub-speciesof bacteria in the sample.

Example 24

A method as recited in Example 19, wherein the microorganism type isdefined as a particular strain of virus and the list of analytecompounds includes a sufficient number of analyte compounds that arediagnostic of the particular viral strain to enable identification ofthe presence or absence of the particular viral strain in the sample.

Example 25

A method as recited in Example 19, wherein the microorganism type isdefined as a particular strain of virus and the list of analytecompounds includes a sufficient number of analyte compounds that arediagnostic of the particular viral strain to enable identification ofthe presence or absence of the particular viral strain in the sample.

Example 26

A method for identifying the presence or absence of a protein orpolypeptide analyte compound within a sample comprising a mixture ofcompounds that includes a plurality of protein compounds or a pluralityof polypeptide compounds or pluralities of both protein and polypeptidecompounds, the method comprising:

-   -   (a) introducing a portion of the liquid sample into an        electrospray ionization source of a mass spectrometer;    -   (b) forming positively charged ions of the mixture of compounds        of the portion of the liquid sample by electrospray ionization,        the positively charged ions comprising a plurality of        first-generation ion species;    -   (c) isolating a plurality of subsets of the first-generation ion        species comprising respective mass-to-charge (m/z) ratio ranges,        wherein each m/z ratio range includes an m/z ratio of an ion        species comprising a respective protonation state of the analyte        compound;    -   (d) generating a plurality of first-generation product ion        species from the isolated plurality of subsets of the        first-generation ion species by causing the isolated plurality        of subsets of the first-generation ion species to be reacted,        for a predetermined time duration, with reagent anions that,        upon reaction, extract protons from each ion species that        comprises a respective protonation state of the analyte        compound;    -   (e) generating a mass spectrum of the first-generation product        ion species; and    -   (f) identifying either the presence of the analyte compound        within the sample if the mass spectrum comprises one or more        lines at respective predetermined m/z ratios that comprise        respective intensities above a predetermined threshold or the        absence of the analyte compound within the sample otherwise.

Example 27

A method as recited in Example 26, further comprising repeatedlyperforming steps (a) through (f) a plurality of times, wherein eachrepetition of step (a) comprises introducing, into the electrosprayionization source, an eluate from a chromatographic column correspondingto a respective retention time.

Example 28

A method as recited in Example 26, wherein the step (f) furthercomprises determining, if the mass spectrum comprises one or more linesat respective predetermined m/z ratios that comprise respectiveintensities above a predetermined threshold, a quantity or concentrationof the analyte compound within the sample based on the one or moreintensities.

Example 29

A method as recited in Example 26, further comprising, after the step(b) of forming positively charged ions and prior to the step (c) ofisolating a plurality of subsets of the first-generation ion species,the steps of:

-   -   (b1) isolating a subset of the first-generation ion species        comprising a randomly-selected mass-to-charge (m/z) ratio range;    -   (b2) generating a plurality of product ion species from the        isolated subsets of the first-generation ion species by causing        the isolated subset of the first-generation ion species to be        reacted with reagent anions that, upon reaction, extract protons        from each ion species that comprises a respective protonation        state of the analyte compound or a respective protonation state        of another protein or polypeptide compound;    -   (b3) generating a mass spectrum of the product ion species; and    -   (b4) automatically determining the m/z ratio ranges to be used        in the subsequent step (c), based on the mass spectrum of the        product ions.

Example 30

A method as recited in Example 28, wherein the step (b4) comprisesautomatically determining, from the mass spectrum, a set of m/z ratioscorresponding to multiply-protonated ion species of the other protein orpolypeptide compound.

Example 31

A method of identifying the presence of absence of a microorganism in asample, comprising:

-   -   making an extract of the sample;    -   repeatedly executing the method recited in Example 26 so as to,        at each execution, identify the presence or absence of a        different respective protein or polypeptide analyte compound        within the sample extract; and    -   identifying the presence of the microorganism within the sample        if the presence of each respective protein or polypeptide        analyte compound within the sample extract or the absence of the        microorganism within the sample otherwise.

CONCLUSIONS

The use of PTR-type of ion-ion reactions as taught in this document hasseveral advantages for analysis of complex mixtures of protein orpolypeptide ions. A first significant advantage is provided by thegreatly improved signal-to-noise ratio as may be readily observed bycomparing FIG. 3 with FIG. 4. Even though some charge is lost (i.e.,complete neutralization) as a result of the PTR process, a significantsignal-to-noise ratio is gained as a result of the reaction ofmultiply-charged proteins with singly charged anions. The rate of such areaction is proportional to the square of the product of the charges.Thus, the originally highly-charged analyte ions are converted intoless-charged PTR product ions whose mass spectral signatures appear atsignificantly greater mass-to-charge ratios. By contrast,low-charge-state chemical background ions are less significantlyaffected by the PTR process during a typical experimental reactionperiod because of the low rates of reaction of such ions. This processessentially removes the mass spectral signatures of the proteins andpolypeptides from the low-mass, low-charge-state chemical background“noise”. For example, as shown in FIG. 4, the background ions arerepresented by the large singly-charged peak that is “left behind” atm/z≈642. It is also believed that adducts or water molecules stilladhered to large proteins are removed as a result of the exothermic heatof reaction (at least 125 kcal/mol) deposited by the PTR reaction. Thetransformation of such ions into simple protonated molecules may furtherenhance signal-to-noise characteristics. Potentially, the number ofprotein identifications obtained via this approach could exceed currentcomplex top-down methods utilizing some form of separation technology.

A second important advantage associated with methods in accordance withthe present teachings is provided by greatly improved charge stateassignment. For example, the inventors have experimentally determinedthat approximately 75% of the charge state assignments for individualcharge states may be correctly assigned by employing methods inaccordance with the present teachings. This improved ability torecognize charge states results from the significantly improvedsignal-to-noise ratio. In turn, this provides more accuratedetermination of the molecular weight of the protein or polypeptide.This comparison applies to the current Patterson-FFT charge statealgorithm that is frequently used for real-time charge statedetermination. Another important advantage associated with methods inaccordance with the present teachings is provided by the ability toperform rapid throughput analyses. When combined with the Fast PartialChromatographic Separation technique applied above, these methods allowfor analyses of samples in a high throughput fashion on a time scale ofone minute or less.

The discussion included in this application is intended to serve as abasic description. Although the invention has been described inaccordance with the various embodiments shown and described, one ofordinary skill in the art will readily recognize that there could bevariations to the embodiments and those variations would be within thespirit and scope of the present invention. Thus, the reader should beaware that the specific discussion may not explicitly describe allembodiments possible; many alternatives are implicit. Accordingly, manymodifications may be made by one of ordinary skill in the art withoutdeparting from the scope of the invention as described by the claims.Neither the description nor the terminology is intended to limit thescope of the invention. Any patents, patent applications, patentapplication publications or other literature mentioned herein are herebyincorporated by reference herein in their respective entirety as iffully set forth herein.

1. A method for identifying the presence or absence of a protein orpolypeptide analyte compound within a liquid sample comprising a mixtureof compounds that includes a plurality of protein compounds or aplurality of polypeptide compounds or pluralities of both protein andpolypeptide compounds, the method comprising: (a) introducing a portionof the liquid sample into an electrospray ionization source of a massspectrometer; (b) forming positively charged ions of the mixture ofcompounds of the portion of the liquid sample by electrosprayionization, the positively charged ions comprising a plurality of ionspecies; (c) isolating a first subset of the ion species comprising afirst mass-to-charge (m/z) ratio range that includes an m/z ratio of amultiply-protonated molecular species of the analyte compound; (d)generating a plurality of first-generation product ion species from theisolated first subset of ion species by causing the isolated firstsubset of ion species to be reacted, for a predetermined time duration,with reagent anions that, upon reaction, extract protons from each ofone or more ion species that comprises a protonated molecular species ofa protein or polypeptide compound; (e) generating mass spectrum data,using a mass analyzer, of either the first-generation product ionspecies or of second-generation product ion species generated from thefirst-generation product ion species; (f) identifying one or moresequences of mass spectral lines of the mass spectrum by performingautomatic real-time deconvolution of the mass spectrum data, whereineach said sequence of lines corresponds to a charge-state sequence ofions corresponding to a respective protein or polypeptide compound ofthe liquid sample; and (g) identifying the presence of the analytecompound within the sample if an identified sequence of mass spectrallines comprises a sequence of m/z ratios that is diagnostic of theprotein or polypeptide analyte compound.
 2. A method as recited in claim1, further comprising repeating the steps (a) through (e) a second time,wherein the steps (f) and (g) are performed during or prior to thesecond performing of the steps (a) through (e).
 3. A method as recitedin claim 1, further comprising repeatedly performing steps (a) through(g) a plurality of times, wherein each repetition of step (a) comprisesintroducing, into the electrospray ionization source, an eluate from achromatographic column corresponding to a respective retention time. 4.(canceled)
 5. A method as recited in claim 1, wherein: the step (c)comprises further isolating a second subset of the ion speciescomprising a second m/z ratio range that includes an m/z ratio of amultiply-protonated molecular species of a second protein or polypeptideanalyte compound; and the step (g) comprises identifying the presence ofthe second analyte compound within the sample if another identifiedsequence of mass spectral lines comprises a sequence of m/z ratios thatis diagnostic of the second protein or polypeptide analyte compound. 6.A method as recited in claim 1, wherein the step (g) further comprisesidentifying the presence of a second protein or polypeptide analytecompound within the sample if another of the one or more identifiedsequences of mass spectral lines comprises a sequence of m/z ratios thatis diagnostic of the second protein or polypeptide anal to compound. 7.A method as recited in claim 5, wherein the step (c) comprisessimultaneously isolating the first subset of the ion species comprisingthe first m/z ratio and the second subset of the ion species comprisingthe second m/z ratio range such that the first and second m/z ratioranges are non-contiguous.
 8. A method as recited in claim 1, whereinthe step (d) of generating a plurality of first-generation product ionspecies comprises causing the isolated first subset of ion species andreagent anions to be reacted for a time duration that causes the production species to be stable against decomposition during the subsequentgeneration of the mass spectrum in step (e).
 9. (canceled)
 10. A methodas recited in claim 1, wherein the step (d) of generating a plurality offirst-generation product ion species includes applying a supplemental ACvoltage across electrodes of an ion trap within which the isolated firstsubset of ion species are reacted with reagent anions, wherein afrequency of the supplemental AC voltage is such that ion-ion reactionbetween the reagent anions and selected first-generation product ionspecies is partially inhibited.
 11. A method as recited in claim 10,wherein the frequency of the supplemental AC voltage is such that,subsequent to the execution of step (d), product ions formed from theanalyte compound exist substantially as a single ion species having aparticular charge state.
 12. A method as recited in claim 11, wherein:the step (e) comprises generating a mass spectrum of thefirst-generation product ion species; and wherein the mass of the singleion species is greater than 20,000 Da and the charge state of the singleion species is sufficiently great such that ions of the single ionspecies may be detected, during the generation of the mass spectrum, byeither a quadrupole mass analyzer, a Fourier transform ion cyclotronresonance mass spectrometer or an electrostatic trap mass analyzer. 13.A method as recited in claim 1, wherein the step (e) of generating amass spectrum comprises generating a mass spectrum of second-generationproduct ion species, wherein the second-generation product ion speciesare generated by the steps of: isolating a subset of thefirst-generation product ion species comprising a particular product-ionm/z ratio range; and fragmenting the isolated subset of thefirst-generation product ion species so as to form fragment ion species,wherein the fragment ion species comprise the second-generation production species.
 14. A method as recited in claim 1, wherein the step (e) ofgenerating a mass spectrum comprises generating a mass spectrum ofsecond-generation product ion species, wherein the second-generationproduct ion species are generated by: causing the first-generationproduct ion species to be reacted, for a second predetermined timeduration, with the reagent anions, wherein products of reaction betweenthe first-generation product ion species and the reagent anions comprisethe second-generation product ion species.
 15. A method as recited inclaim 14, wherein a supplemental AC voltage is applied across electrodesof an ion trap within which the first-generation product ion species arereacted with the reagent anions, wherein a frequency of the supplementalAC voltage is such that ion-ion reaction between the reagent anions andselected product ion species is inhibited.
 16. A method as recited inclaim 1, further comprising generating the liquid sample comprising themixture of compounds by a procedure comprising: (i) culturingmicroorganisms or cells; (ii) lysing the cultured microorganisms orcells; and (iii) extracting proteins from the lysate of culturedmicroorganisms or cells.
 17. (canceled)
 18. A method of identifying thepresence or absence of a microorganism type in a sample, comprising: (a)identifying a list of analyte compounds whose simultaneous presence inthe sample is diagnostic of the presence of the microorganism type inthe sample, said list of analyte compounds comprising protein compounds,polypeptide compounds or both protein and polypeptide compounds; (b)extracting, from the sample, a liquid solution comprising a mixture ofsample-derived proteins and polypeptides; (c) introducing at least afirst portion of the liquid solution into an ionization source of a massspectrometer; (d) generating, from the at least first portion of theliquid solution at the ionization source, positively charged ions of themixture of compounds, the positively charged ions comprising a pluralityof ion species; (e) isolating at least a first subset of the pluralityof ion species, each isolated subset of the at least a first isolatedsubset comprising a respective mass-to-charge (m/z) ratio range; (f)generating a plurality of first-generation product ion species from eachisolated subset of ion species by causing each said isolated subset ofion species to be reacted, for a predetermined time duration, withreagent anions that, upon reaction, extract protons from each of one ormore ion species of said isolated subset of ion species that comprises aprotonated molecular species of a protein or polypeptide compound; (g)generating at least one mass spectrum, using a mass analyzer of the massspectrometer, either of some or all of the first-generation product ionspecies or of a plurality of second-generation product ion speciesgenerated by further reaction of the first-generation product ionspecies; (h) identifying one or more sequences of mass spectral lines ofeach of the at least one mass spectrum by performing, automaticreal-time deconvolution of data of each said mass spectrum. wherein eachsaid sequence of lines corresponds to a charge-state sequence of ionscorresponding to a respective protein or polypeptide compound of theliquid sample; (i) for each respective analyte compound in the list,identifying the presence of the respective analyte compound within theliquid solution if an identified sequence of mass spectral linescomprises a sequence of m/z ratios that is diagnostic of the saidrespective analyte compound; and (j) identifying the presence of themicroorganism type within the sample if the presence of each and everyanalyte compound of the list of analyte compounds is identified withinthe liquid solution.
 19. A method as recited in claim 18, wherein theleast a first portion of the liquid solution comprises a plurality ofportions of the liquid solution, each portion comprising an eluate froma chromatographic column at a respective retention time and wherein aperforming of the steps (h) and (i) is performed concurrently with aperforming of one or more of the steps (c) through (g).
 20. A method asrecited in claim 18, wherein the microorganism type is defined as aparticular genus of bacteria and the list of analyte compounds includesa sufficient number of analyte compounds that are diagnostic of theparticular genus of bacteria to enable identification of the presence orabsence of the particular genus of bacteria in the sample.
 21. A methodas recited in claim 18, wherein the microorganism type is defined as aparticular species of bacteria and the list of analyte compoundsincludes a sufficient number of analyte compounds that are diagnostic ofthe particular species of bacteria to enable identification of thepresence or absence of the particular species of bacteria in the sample.22. A method as recited in claim 18, wherein the microorganism type isdefined as a particular sub-species of bacteria and the list of analytecompounds includes a sufficient number of analyte compounds that arediagnostic of the particular sub-species of bacteria to enableidentification of the presence or absence of the particular sub-speciesof bacteria in the sample.
 23. A method as recited in claim 18, whereinthe microorganism type is defined as a particular strain or serovar, andthe list of analyte compounds includes a sufficient number of analytecompounds that are diagnostic of the particular strain or serovar toenable identification of the presence or absence of the particularstrain or serovar in the sample.
 24. A method as recited in claim 18,wherein the microorganism type is defined as a particular strain ofvirus and the list of analyte compounds includes a sufficient number ofanalyte compounds that are diagnostic of the particular viral strain toenable identification of the presence or absence of the particular viralstrain in the sample.
 25. A method for identifying the presence orabsence of a protein or polypeptide analyte compound within a samplecomprising a mixture of compounds that includes a plurality of proteincompounds or a plurality of polypeptide compounds or pluralities of bothprotein and polypeptide compounds, the method comprising: (a)introducing a portion of the liquid sample into an electrosprayionization source of a mass spectrometer; (b) forming positively chargedions of the mixture of compounds of the portion of the liquid sample byelectrospray ionization, the positively charged ions comprising aplurality of first-generation ion species; (c) co-isolating a pluralityof subsets of the first-generation ion species, each subset comprising arespective mass-to-charge (m/z) ratio range, wherein the m/z ratioranges are non-contiguous and each m/z ratio range includes an m/z ratioof an ion species comprising a respective protonation state of theanalyte compound; (d) generating a plurality of first-generation production species from the isolated plurality of subsets of thefirst-generation ion species by causing the co-isolated plurality ofsubsets of the first-generation ion species to be reacted, for apredetermined time duration, with reagent anions that, upon reaction,extract protons from each ion species that comprises a respectiveprotonation state of the analyte compound; (e) generating a massspectrum of the first-generation product ion species; and (f)identifying either the presence of the analyte compound within thesample if the mass spectrum comprises one or more lines at respectivepredetermined m/z ratios that comprise respective intensities above apredetermined threshold or the absence of the analyte compound withinthe sample otherwise.
 26. (canceled)
 27. A method as recited claim 25,wherein the step (f) further comprises determining, if the mass spectrumcomprises one or more lines at respective predetermined m/z ratios thatcomprise respective intensities above a predetermined threshold, aquantity or concentration of the analyte compound within the samplebased on the one or more intensities.
 28. A method as recited in claim25, further comprising, after the step (b) of forming positively chargedions and prior to the step (c) of isolating a plurality of subsets ofthe first-generation ion species, the steps of: (b1) isolating a subsetof the first-generation ion species comprising a randomly-selected m/zratio range; (b2) generating a plurality of product ion species from theisolated subsets of the first-generation ion species by causing theisolated subset of the first-generation ion species to be reacted withreagent anions that, upon reaction, extract protons from each ionspecies that comprises a respective protonation state of the analytecompound or a respective protonation state of another protein orpolypeptide compound; (b3) generating a mass spectrum of the product ionspecies; and (b4) automatically determining the m/z ratio ranges to beused in the subsequent step (c), based on the mass spectrum of theproduct ions.
 29. A method as recited in claim 27, wherein the step (b4)comprises automatically determining, from the mass spectrum, a set ofm/z ratios corresponding to multiply-protonated ion species of the otherprotein or polypeptide compound.
 30. (canceled)